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C W Vogel

Publications and source records attributed to C W Vogel.

At least 37 records · Page 2Linked to original sources
PubMed1994

Antibody-mediated complement activation on nucleated cells. A quantitative analysis of the individual reaction steps.

The sequential molecular events of the initiation, amplification, and membrane attack phases of classical C pathway activation on nucleated cells were investigated. As a model system, C-susceptible human melanoma cells (SK-MEL-93-2) expressing the disialoganglioside Ag GD3 were studied. Activation of the classical C pathway was initiated by the anti-GD3 mAb R24 (murine IgG3). The initiation phase is characterized by a very inefficient molar ratio of deposited C1q per Ab molecule. At an Ab density of 5.86 x 10(6) molecules/cell, only 3% of cell-bound R24 molecules form suitable pairs for C1q binding. During the amplification phase maximally 2.44 x 10(6) molecules of C4 and 0.67 x 10(6) molecules of C2/cell are being bound to form the C3 convertase. Despite the rather inefficient binding of C2, the C3 convertase is highly active in depositing high numbers of C3b molecules on the cell surface. Maximum binding of C3b occurred within 5 min of incubation with a total number of 2.1 x 10(7) molecules/cell. This indicates amplification factors at the level of C4 and C3 of 28 (C4/C1q) and 241 (C3/C1q), respectively. C3b was found to be rapidly cleaved into iC3b. As a result of this rapid C3b degradation, the membrane attack phase is initiated with a relatively inefficient C5 activation. The maximal number of 9.5 x 10(5) molecules C5b/cell corresponds to a molar ratio of C5:C3 of only 1:22. The deposition of C5b led to the subsequent maximum binding of the following numbers of molecules of terminal C components per cell: C6, 0.8 x 10(6); C7, 0.89 x 10(6); C8, 0.82 x 10(6); C9, 1.8 x 10(6). These numbers correspond to average molar ratios (calculated per C5b molecule) of C5b/C6/C7/C8/C9 of 1/0.85/0.94/0.86/1.88. In addition to the monomeric C9, dimeric and polymeric (12- to 16-mer) forms of the molecule could be demonstrated. Collectively, our data represent a first comprehensive quantitative analysis of classical pathway activation on a nucleated cell.

Antibodies, Monoclonal↗
PubMed1994

Immunoreactivity and function of oligosaccharides in cobra venom factor.

Cobra venom factor (CVF) is the nontoxic C-activating glycoprotein in cobra venom. It is a structural and functional analogue of complement component C3. Previous work has established that the major oligosaccharide chain of CVF is a symmetric fucosylated biantennary complex-type N-linked chain containing an alpha-galactosylated Le(x) antigenic epitope, Gal alpha 1-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1, a novel carbohydrate structure. We show that naturally occurring anti-alpha-Gal Ab present in normal human serum binds to CVF. In view of a possible human application of CVF, we studied the effect of this anti-alpha-Gal Ab on CVF function as well as the effect of oligosaccharide modification or removal on CVF activity and immunoreactivity with the anti-alpha-Gal Ab. The immunoreactivity of CVF with the anti-alpha-Gal Ab is abolished upon de-alpha-galactosylation or complete deglycosylation. De-alpha-galactosylated CVF and deglycosylated CVF were found to be equally active in forming a stable C3/C5 convertase with human factor B and in decomplementing human serum indicating that the oligosaccharide chains of CVF are not required for its C-activating function. Consistent with the unaltered functional activity, deglycosylation of the molecule caused only minor changes in secondary structure as judged by far UV circular dichroism analysis. However, the oligosaccharide chains appear to contribute to the thermal stability of CVF, because deglycosylation caused the molecule to be more sensitive to temperature. Inasmuch as rCVF produced in mammalian cells can be expected to contain sialylated oligosaccharide chains, we also prepared sialylated CVF by enzymatic sialylation of de-alpha-galactosylated and de-alpha-fucosylated CVF with 2,6-sialyltransferase. It was found that the secondary structure and the activity of sialylated CVF were unchanged compared to native CVF.

Animals↗
PubMed1993

Delivery of radionuclides to pretargeted monoclonal antibodies using dihydrofolate reductase and methotrexate in an affinity system.

A novel affinity system for a two-phase delivery of radionuclides to tumor cells has been developed. In the first phase, a nontoxic bivalent monoclonal antibody conjugated to an enzyme is targeted to the tumor cells. In the second phase, a radionuclide-derivatized enzyme inhibitor, specific for the enzyme conjugated to the antibody, is administered. The model system selected for this study is the recombinant human enzyme dihydrofolate reductase (rhDHFR) and its high-affinity competitive inhibitor methotrexate (MTX). MTX was labeled with a radionuclide by covalent attachment of diethylenetriaminepentaacetic acid (DTPA) complexed with 111In. Using the gamma-carboxyl residue of MTX for the attachment of DTPA, binding of the inhibitor to rhDHFR was not affected. The inhibitory activities of nonderivatized MTX and DTPA-MTX were indistinguishable. Human K562 erythroleukemia cells were used to evaluate under in vitro conditions the DHFR-MTX affinity system for the delivery of 111In-labeled DTPA-MTX to pretargeted alpha-transferrin receptor antibody-rhDHFR conjugates (alpha-TFR-DHFR). The data demonstrate that the delivery of 111In is dose dependent and highly specific. Under saturating conditions, binding of 111In-DTPA-MTX to alpha-TFR-DHFR-treated cells was 14-fold higher than to cells treated with nonconjugated alpha-TFR antibody. Further experiments indicated that the low level of nonspecific binding of 111In-DTPA-MTX was comparable to that of 111In-DTPA, known for its complete extracellular distribution and rapid clearance through the kidneys. Based on the data of this study, antibody-conjugated rhDHFR and radionuclide-labeled DTPA-MTX complexes provide components for an alternative radioimmunotherapeutic approach that can be expected to result in improved tumor tissue ratios of both the targeting moiety and the radionuclide-labeled derivative as compared to current approaches.

Antibodies, Monoclonal↗
PubMed1993

Molecular basis of complement resistance of human melanoma cells expressing the C3-cleaving membrane protease p65.

The molecular mechanism of complement resistance of the human SK-MEL-170 melanoma cell line was investigated. The cells have been shown to express the C3b-cleaving membrane protease p65. To delineate the molecular consequences of the C3b-cleaving activity for the complement cytotoxicity, the molecular events during the initiation (R24 monoclonal antibody, C1), amplification (C4, C3), and membrane attack (C5, C9) phases of complement were studied in comparison to a complement-susceptible human melanoma line (SK-MEL-93-2). No cleavage of C4b and C5b, 2 molecules structurally similar to C3b, was observed on the cells during classical pathway activation indicating the specificity of the p65 protease for the C3b molecule. The rapid degradation of C3b by p65 on the surface of complement-resistant SK-MEL-170 cells generates a M(r) 30,000 C3 alpha'-chain-fragment detectable as early as 1 min after complement activation, whereas no such fragment was present in detectable amounts on complement-susceptible cells. As a result of the rapid C3b proteolysis by p65 on resistant SK-MEL-170 cells, less C5 convertases are formed, which in turn results in the formation of a lower number of terminal complement components and membrane attack complexes. R24 antibody and C1q binding to the resistant cells was slightly lower as to susceptible cells. C4 binding studies, however, revealed that the observed difference in antibody and C1q binding has no influence on the complement resistance of SK-MEL-170 cells: significantly more C4b was bound to complement-resistant (1565 +/- 92 fg/cell) as compared to susceptible cells (715 +/- 31 fg/cell). On extraction of the molecular forms of C4 bound to the cell membranes, an additional high molecular weight C4 species--apparently a C4b-C4b homodimer--appeared only on the resistant SK-MEL-170 cells that may function as a residual back-up C5 convertase. Collectively, these results show that SK-MEL-170 human melanoma cells evade complement-mediated cytolysis despite sufficient activation of early components of the classical complement pathway by p65-mediated rapid degradation of surface-bound C3b, leading to a significant reduction in membrane attack complex formation. Thus, rapid cleavage of surface deposited C3b was established as a powerful mechanism of complement resistance.

Animals↗
PubMed1992

Primary structure of cobra complement component C3.

Complement component C3 is a multifunctional protein known to interact specifically with more than 10 different plasma proteins or cell surface receptors. Cobra venom contains cobra venom factor, a structural analogue of C3 that shares some properties with C3 (e.g., formation of a C3/C5 convertase) but differs in others (e.g., susceptibility to regulation by factors H and I). The elucidation of structural differences between C3 and cobra venom factor can be expected to help identify functionally important regions of C3 molecules. To that end we have undertaken the molecular cloning of both cobra C3 and cobra venom factor to take advantage of the unique biologic system where both proteins are produced by the same species. We report the primary structure of cobra C3 mRNA and the derived protein structure. Cobra C3 mRNA is 5211 bp in length. It contains an open reading frame of 4953 bp coding for a single pre-pro-C3 molecule, consisting of a 22-amino acid signal sequence, a 633-amino acid beta-chain (70 kDa), and a 992-amino acid alpha-chain (112 kDa) which is separated from the beta-chain by four arginine residues. There are no N-glycosylation sites in cobra C3. Cobra C3 exhibits approximately 58% nucleotide sequence identity with C3 from mammalian species. At the protein level, sequence identity is approximately 52% and sequence similarity approximately 71%. All 27 cysteine residues are highly conserved as are the C3 convertase cleavage site, the thioester site, and the factor B binding site. Cobra C3 also seems to have homologous binding sites for factor H and properdin, as well as a conserved sequence in the functionally important region of the C3a anaphylatoxin. The sequence homology at the CR2 and CR3 binding sites does not exceed the overall sequence homology. Accordingly, the existence of CR2 and CR3 binding sites can neither be deduced nor excluded.

Amino Acid Sequence↗
PubMed1992

Structure of the major oligosaccharide of cobra venom factor.

Cobra venom factor (CVF), the complement-activating glycoprotein in cobra venom, contains three or possibly four N-linked oligosaccharide chains per molecule and is devoid of O-linked saccharides. Analysis by lectin-affinity staining revealed the presence of complex-type oligosaccharides containing non-reducing terminal alpha-galactosyl residues and fucose residues linked to the proximal N-acetylglucosamine. Sialic acid residues could not be detected. For their structural analysis, the oligosaccharides were released by hydrazinolysis and fractionated on Bio-Gel P-4. Approximately 80% of the eluted oligosaccharides have a size equivalent of 17 +/- 2 glucose units. The major oligosaccharide representing about 45% of the total carbohydrate present in CVF was purified to homogeneity by MicroPak AX-5 HPLC and its structure was analyzed by sequential exoglycosidase digestion. The positions of the glycosidic linkages of the sugar residues were established by methylation analysis of CVF-derived glycopeptides. The data of these analyses indicated that the major oligosaccharide has a symmetrical fucosylated biantennary complex-type structure terminating with unusual alpha-galactosyl residues.

Animals↗
PubMed1991

Novel trifunctional carrier molecule for the fluorescent labeling of haptens.

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

A carbohydrate-directed heterobifunctional cross-linking reagent for the synthesis of immunoconjugates.

A novel, highly water-soluble, heterobifunctional cross-linking reagent, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), was synthesized which contains a hydrazide moiety for coupling to aldehyde groups generated in the carbohydrate residues of antibodies by mild periodate oxidation, and a pyridyl disulfide moiety for coupling to molecules with a free sulfhydryl group. Since the carbohydrate moieties are distal to the antigen binding region of antibodies, derivatization with this cross-linker minimizes impairment of the antigen binding function. Derivatization of the human monoclonal IgM antibody 16-88 against human colon carcinoma cells with as many as 16 TPCH cross-linker molecules did not impair its antigen binding capability. Using mild oxidation conditions for antibody derivatization, sialic acid residues were identified as attachment sites for the cross-linker molecules, since after desialylation of antibody 16-88 by neuraminidase virtually no cross-linker molecules could be incorporated. Comparison of TPCH with S-(2-thiopyridyl)mercaptopropionic acid hydrazide and S-(2-thiopyridyl)-L-cysteine, two related cross-linking reagents, revealed that TPCH is most efficiently incorporated into periodate-treated antibody. Based on the structural differences of the cross-linkers, the more efficient incorporation of TPCH appears to be a function of the presence of a hydrazide moiety with an adjacent amino group. When three to four molecules of pyridyl disulfide-derivatized barley toxin were coupled to TPCH-derivatized antibody 16-88, the antigen binding capability remained uncompromised. In addition, no significant impairment of toxin activity upon coupling to the antibody was observed. Based on these data, TPCH may be very useful for the synthesis of immuno-conjugates with no or only minimal impairment of the antigen binding function.

Antibodies, Monoclonal↗
PubMed1990

A continuous flow immunoassay for rapid and sensitive detection of small molecules.

An immunosensor operating in continuous flow and capable of detecting low molecular weight antigens is described. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of additional reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within 3 min in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. Signal detection occurs downstream of the antigen recognition event. In this study, the hapten 2,4-dinitrophenol (DNP) as DNP-lysine was used as model antigen. To generate a labeled antigen, DNP was coupled to the terminal amino group of insulin A chain (tetra S-sulfonate form) which provides two tyrosine residues for the introduction of an 125I-label (DNP-Ins-125I) or three carboxyl groups for the attachment of three fluorescein residues (DNP-Ins-Fl). The radiolabeled antigen was used to establish assay conditions. Subsequently, fluorescein was substituted for the radioisotope label in order to develop an assay independent of the restrictions associated with isotopes. Using this flow immunoassay, we were able to detect DNP-lysine down to a detection limit of 143 nM (29 pmol/200 microliters) using DNP-Ins-125I or DNP-Ins-Fl as labeled antigen. The density of immobilized antibody and the flow rate were identified to be critical parameters for the sensitivity of the assay.

Animals↗
PubMed1990

C3-cleaving membrane proteinase. A new complement regulatory protein of human melanoma cells.

Human melanoma cells resistant to killing by the R24 mAb and human complement rapidly degrade surface-deposited C3b (M. Panneerselvam, S. Welt, L. J. Old, C.-W. Vogel. 1986. J. Immunol. 136:2534). We report that C-resistant melanoma cells express a membrane proteinase that can cleave C3b, generating a cleavage product with a molecular mass of approximately 30 kDa. The C3-cleaving proteinase was identified on the melanoma cells by its cross-reaction with antiserum to p57, a C3-cleaving proteinase previously isolated from human E membranes (C. Charriaut-Marlangue, M. Barel, R. Frade. 1986. Biochem. Biophys. Res. Commun. 140:1113). Preincubation of the C-resistant melanoma cells with anti-p57 IgG or their F(ab')2 fragments increased their susceptibility to complement killing from 25% to approximately 50% and reduced the rate of C3b cleavage and the amount of the 30-kDa fragment generated on the cells. Anti-p57 IgG stained C-resistant melanoma cells by indirect immunofluorescence and precipitated a protein with an apparent molecular mass of 65 kDa. This membrane protein, termed p65, was not detectable on C-susceptible melanoma cells. Membrane extracts from C-resistant melanoma cells also showed C3-cleaving activity when incubated with purified C3 or C3b, similarly generating a C3 fragment of approximately 35 kDa. This fluid-phase C3 cleaving activity could be partially inhibited by anti-p57 IgG. These data suggest that p65 is a C3-cleaving proteinase, antigenically related to p57, that is expressed on C-resistant melanoma cells and responsible for the C resistance of these cells. We propose that the membrane-bound C3-cleaving proteinase represents another C regulatory protein protecting host cells against killing by C.

Blotting, Western↗
PubMed1990

Complement killing of human neuroblastoma cells: a cytotoxic monoclonal antibody and its F(ab')2-cobra venom factor conjugate are equally cytotoxic.

Only a few monoclonal antibodies mediate complement lysis of tumor cells, but for several antibodies it has been demonstrated that a complement-activating function can be introduced by covalent coupling of cobra venom factor (CVF), a non-toxic glycoprotein which is a structural and functional homologue of human complement component C3. In this study we compared the efficacy of complement killing of human neuroblastoma cells by the complement-activating monoclonal antibody 3F8 directed against the GD2 ganglioside antigen with that of its F(ab')2-CVF conjugate. At equal numbers bound per cell the 3F8 antibody and the 3F8 F(ab')2-CVF conjugate were found to be equally cytotoxic in the presence of complement from several species including human. Maximal killing reached up to 98%. The kinetics of killing and the bivalent metal requirement confirmed that the cytotoxic activity of the 3F8 antibody is mediated via the classical pathway and that of the 3F8 F(ab')2-CVF conjugate via the alternative pathway. To achieve a comparable degree of killing, an approximately eight-fold higher concentration of the 3F8 F(ab')2-CVF conjugate was required which appears to be a consequence of the approximately eight-fold lower binding activity of the 3F8 F(ab')2-CVF conjugate compared to the intact 3F8 antibody. Our data suggest that the coupling of CVF to non-cytotoxic antibodies allows the generation of conjugates with a cytotoxic activity similar to that of inherently cytotoxic antibodies.

Antibodies, Monoclonal↗
PubMed1989

The oligosaccharide chains of cobra venom factor are required for complement activation.

To examine the function of the carbohydrate chains of cobra venom factor (CVF), the molecule was enzymatically deglycosylated under non-denaturing conditions with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase). The deglycosylation of CVF chains seems to proceed independently of each other, leading to partially deglycosylated intermediates. Complete deglycosylation of CVF was found to abolish the activity of CVF. The deglycosylated molecule is unable to activate the alternative pathway of complement. Deglycosylated CVF no longer consumes the serum complement activity, it does not induce C3 activation in serum, nor does it induce complement-mediated hemolysis. These results indicate that the carbohydrate moieties of CVF are essential for its role in complement activation.

Animals↗
PubMed1988

A novel cleavage product of human complement component C3 with structural and functional properties of cobra venom factor.

The generation of two cleavage products of human C3, termed C3o and C3p, by incubation with a C3-cleaving protease isolated from cobra venom (Naja naja siamensis) is described. The venom protease removes the C3p fragment (Mr approximately 33,000) from the C3dg region of the C3 alpha-chain. The major cleavage fragment C3o (Mr approximately 140,000) contains the unaltered beta-chain of C3 and two alpha-chain-derived polypeptides of Mr approximately 29,000 and Mr approximately 38,000, respectively. Amino-terminal amino acids sequence analysis of C3p and the three chains of C3o allowed the identification of the exact location of the two alpha-chain-derived fragments of C3o and the three cleavage sites of the venom protease. The chain structure of C3o resembles those of C3c and cobra venom factor. In contrast to C3c but like cobra venom factor (and C3b), C3o was found to support the activation of the serine protease Factor B by cleavage in the presence of Factor D and Mg2+ into Bb and Ba, generating an enzymatically active complex that is able to cleave a fluorogenic peptide substrate for C3 convertases. Since the only stretch of amino acid residues of C3o not present in C3c is the carboxyl terminus of the Mr approximately 29,000 chain of C3o, it is suggested that this region is important for the interaction with Factor B and convertase formation.

Amino Acid Sequence↗
PubMed1988

Differences in uptake of mycobacteria by human monocytes: a role for complement.

We investigated the influence of serum factors on the uptake of various species of mycobacteria by human peripheral blood monocytes (PBM). On the basis of the percentage of PBM involved during in vitro uptake, the mycobacteria were of two distinct groups. The mycobacteria of one group, which consisted of Mycobacterium avium complex and M. chelonae, were taken up by many PBM; the other group, consisting of M. tuberculosis, M. kansasii, M. fortuitum, and M. gordonae, were taken up by fewer PBM. M. scrofulaceum was intermediate to these two groups on the basis of its uptake by PBM. Serum depleted of complement by heating or treatment with cobra venom factor significantly reduced the extent of PBM involvement with M. avium complex, indicating that complement is an important serum component mediating the uptake of M. avium complex organisms. Preincubation of mycobacteria with serum containing 10 mM EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and 10 mM MgCl2 resulted in uptake by a high percentage of PBM, while preincubation in heated serum or serum containing 10 mM EDTA resulted in a significantly reduced percentage of PBM involved in uptake of M. avium complex organisms, indicating that these organisms are activators of the alternative pathway of complement. Incubation of M. avium complex organisms in human serum consumed 51% of the hemolytic complement activity. Parallel experiments indicated that serum had a lesser effect on the uptake of M. tuberculosis. Thus, serum is important in in vitro M. avium complex uptake by PBM; complement has a major role in the effect of serum, but this role is less important with M. tuberculosis.

Adolescent↗
PubMed1987

Antibody conjugates with cobra venom factor. Synthesis and biochemical characterization.

Immunoconjugates are semi-synthetic hybrid proteins which bear great promise to become a new generation of anti-tumor agents. While many immunoconjugates have been shown to be selectively cytotoxic in in vitro model systems, dramatic in vivo anti-tumor effects have not been reported. To improve the activity of immunoconjugates, careful structure-function analyses have to be performed. We report here such an analysis for immunoconjugates consisting of a monoclonal anti-tumor antibody (MoAb) and cobra venom factor (CVF), the complement-activating glycoprotein from cobra venom, synthesized with the heterobifunctional crosslinking reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). It is shown that a reaction mixture after protein coupling contains free MoAb and CVF as well as hybrid proteins of different compositions (dimers (MoAb-CVF), trimers (MoAb2-CVF, MoAb-CVF2), tetramers (MoAb-CVF3, MoAb2-CVF2, MoAb3-CVF), and some higher oligomers). While free MoAb and CVF can be removed by size exclusion chromatography, separation of different oligomeric hybrid proteins is not possible by this method. From the biochemical characterization of the hybrid proteins, which included the determination of sedimentation coefficients, recording of circular dichroism spectra with subsequent determination of secondary structure, and ultrastructural analysis by transmission electron microscopy, it was concluded that the two proteins do not undergo major structural changes upon coupling, and that the coupling of the two proteins is random with no preferential relative orientation. The functional inactivation of CVF was substantial (approximately 70%) due to both derivatization with SPDP and subsequent conjugation to the MoAb, with conjugation being relatively more inactivating than derivatization. In contrast, the binding activity of the antibody was far less susceptible to inactivation. In conclusion, our data indicate that immunoconjugate synthesis with heterobifunctional crosslinking reagents results in a mixture of heterogeneous hybrid proteins and causes substantial functional inactivation. For successful in vivo anti-tumor activity of future immunoconjugates with CVF and other protein ligands better methods for immunoconjugate synthesis will have to be developed.

Antibodies, Neoplasm↗
PubMed1987

Resistance of human melanoma cells against the cytotoxic and complement-enhancing activities of doxorubicin.

The anticancer agent doxorubicin has two different effects on human SK-MEL-170 melanoma cells: the well known direct cytotoxicity and a marked enhancement of their susceptibility to killing by the R24 monoclonal anti-GD3) ganglioside antibody and human complement. This complement-enhancing effect of doxorubicin is also present after covalent immobilization onto glycerol-coated glass beads preventing cellular uptake of the drug (M. Panneerselvam, R. Bredehorst, and C-W. Vogel, Proc. Natl. Acad. Sci. USA, 83: 9144-9148, 1986). In order to investigate the effect of doxorubicin resistance on the complement-enhancing activity of the drug, we have established a doxorubicin-resistant SK-MEL-170 subline. The development of drug resistance in these melanoma cells was associated with multiple phenotypical changes including an increased expression of at least four high molecular weight plasma membrane proteins or glycoproteins with molecular weights of approximately 220,000, 180,000, 150,000, and 130,000, respectively. The drug-resistant cells accumulated doxorubicin at approximately 2-fold lower amounts in accordance with a 2-fold higher efflux of doxorubicin from these cells. The basal complement susceptibility of the doxorubicin-resistant cells was reduced by more than 60% presumably as a result of the observed reduced expression of GD3 sites and decreased binding of C3b. Most importantly, the doxorubicin-resistant cells were also resistant against the complement-enhancing effect of the free and immobilized drug. The two doxorubicin resistance phenomena, the resistance to the cytotoxic and to the complement-enhancing activities of the drug, seem to be fundamentally different: (a) to achieve comparable cytotoxic and complement-enhancing effects on drug-sensitive and -resistant SK-MEL-170 cells, the drug-resistant cells required 178-fold higher concentrations of free doxorubicin for the cytotoxic effect but only 5-fold higher amounts of the free drug and 12-fold higher amounts of the immobilized drug for the complement-enhancing effect; (b) resistance against the cytotoxic activity of doxorubicin is associated with reduced intracellular drug accumulation, while the data obtained with immobilized doxorubicin indicate that resistance to the complement-enhancing activity of the drug is independent of cellular drug uptake. These data suggest that different mechanisms are responsible for the two resistance phenomena in doxorubicin-resistant melanoma cells.

Antibodies, Monoclonal↗
PubMed1987

Cobra venom factor and human C3 share carbohydrate antigenic determinants.

As tools to study structural relationships of cobra venom factor (CVF) and human complement component C3, murine monoclonal antibodies to CVF were produced. In this paper we describe two of these monoclonal anti-CVF antibodies designated GV1.8 and GV1.10, both of which bind to carbohydrate epitopes. On immunoblotting, antibody GV1.8 binds to both the alpha- and beta-chains of CVF, whereas antibody GV1.10 binds only to the alpha-chain of CVF. After enzymatic deglycosylation of CVF with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase), both antibodies lose their ability to bind to the deglycosylated protein. Additionally, the free oligosaccharide chains of CVF are able to inhibit the binding of antibodies GV1.8 and GV1.10 to CVF on enzyme-linked immunosorbent assay, further demonstrating their carbohydrate specificity. Both monoclonal antibodies to CVF cross-react with human C3. Antibody GV1.8 binds to both chains of human C3 indicating that the shared antigenic epitope present on the two glycosylated chains of CVF is also present on the two chains of human C3. Antibody GV1.10 cross-reacts only with the beta-chain of human C3 which is the homologous chain to the alpha-chain of CVF. After enzymatic deglycosylation of human C3 by N-glycanase, both antibodies lose their ability to bind to the deglycosylated protein consistent with the carbohydrate nature of the recognized epitopes. These results indicate that CVF and human C3 share carbohydrate epitopes on their homologous and nonhomologous chains.

Carbohydrates↗