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Biomedical subjects

S Canevari

Publications and source records attributed to S Canevari.

At least 109 records · Page 6Linked to original sources
PubMed1986

Carbohydrate epitope defined by an antitumor monoclonal antibody detected on glycoproteins and a glycolipid by immunoblotting.

The murine monoclonal antibody (MAb) MOv2 was found to be directed against the carbohydrate moieties of different kinds of molecules expressed on a human ovarian cystoadenocarcinoma. To define further the glycoconjugates carrying the MOv2-defined epitope, different procedures were used to analyze materials from surgical specimens and carcinoma cell lines. SDS-PAGE and immunoblotting showed glycoprotein molecules migrating in the gel as high and intermediate molecular weight components. A low-molecular-weight band, migrating approximately with the dye front, was also immunostained by MOv2. On the other hand, the immunostaining of high-performance thin-layer chromatography (HPTLC) of the total glycolipid extract and its neutral and acid fractions, after DEAE chromatography, showed selective reactivity with a neutral glycolipid. Reanalysis by immunoblotting of this glycolipid band scraped off the HPTLC plate indicated that it corresponds to the low-molecular-weight component. Periodate oxidation and Pronase digestion further demonstrate the saccharide nature of the determinant on both types of glycoconjugates. In conclusion, we report evidence that with a single analytical procedure, i.e., immunoblotting, it is possible to recognize the same carbohydrate determinant carried on both protein and lipid molecules.

Antibodies, Monoclonal↗
PubMed1985

Ricin A chain conjugated with monoclonal antibodies selectively killing human carcinoma cells in vitro.

Ricin A chain was coupled to murine monoclonal antibodies MBr1 and MOv2 respectively raised against human breast and ovarian carcinomas. Inhibition of protein synthesis only occurred in those cultured human tumor cells bearing the appropriate target antigens, demonstrating that both components of the conjugate were unchanged in regards to specificity and toxicity. Conjugates were 125-200 times more efficient in inhibiting [3H]proline incorporation than the uncoupled ricin A chain. They were however unable to kill the entire population of the appropriate cells even after repeated treatment. Although the two monoclonal antibodies had similar binding kinetics, the conjugates differed in their cytotoxicity kinetics. The MBr1-ricin A chain conjugate had slow kinetics, and about 20 hours were needed to obtain a protein synthesis inhibition above 50% on the appropriate line (mammary carcinoma MCF-7). In contrast, the MOv2-ricin A chain conjugate showed very fast kinetics, reaching 50% inhibition after only 30 minutes of treatment on both appropriate cell lines SW626 and HT-29 from ovarian and colon carcinomas, respectively. Growth conditions of cell lines, i.e., adherent cells versus suspended cells, and plating time were found to greatly influence the conjugates' killing efficiencies. These studies confirm the possibility of preparing ricin A chain-antibody conjugates, which retain specific cytotoxicity against tumor cells; but they also underline the need for further in vitro studies of various parameters before one considers a therapeutic use of such conjugates.

Antibodies, Monoclonal↗
PubMed1985

Biochemical analysis of human ovarian cancer-associated antigens defined by murine monoclonal antibodies.

Two monoclonal antibodies (MOv1 and MOv2) raised against a membrane preparation of a human surgical specimen from a mucinous ovarian cystoadenocarcinoma were used to biochemically define their target antigens. The heating of peritumoral mucus-soluble extracts and the sialidase treatment of crude membrane preparations did not affect the binding capacity of MOv1 and MOv2, which, on the contrary, was significantly reduced by periodate oxidation of the same materials. Pronase digestion completely solubilized MOv1-defined antigens, whereas MOv2-defined antigens were only partially solubilized. This, however, did not affect antibody binding with digested products. These data suggest that carbohydrate residues of recognized molecules constitute the antigenic determinants and that sialic acid residues are not involved. Gel filtration on Sepharose 4B of the peritumoral mucus, solubilized either by 200 mM NaCl or Pronase, revealed that most of the antigenic activity eluted in the void-volume fractions with a high carbohydrate content and in the included fractions before the elution volume of the ferritin standard protein. When CsCl gradient equilibrium ultracentrifugation of the solubilized mucus was used, MOv1-recognized antigens sedimented with a density of 1.45 g/ml, while the MOv2-defined epitope was carried by molecules with a density of 1.52 g/ml as well as by molecules with a lower density. Using thin-layer chromatography of organic solvent extracts obtained from mucus and crude membrane preparations, only MOv2-positive molecules could be resolved as a single band of glycolipid. Altogether, these data suggest that the antigens detected by MOv1 are mainly mucins whereas the determinant recognized by MOv2 is carried by both mucins and a glycolipid. To analyze the diagnostic potential of MOv1- and MOv2-recognized molecules, we tested their presence, as soluble products, in supernatants of tumor cell lines and in peritoneal effusions from cancer patients. To this aim, we developed an immunoradiometric assay using the same monoclonal antibody in insolubilized and soluble form. Whereas MOv1-immunoradiometric assay was always negative, by MOv2-immunoradiometric assay it was possible to detect the relevant antigen in 8 of the 10 effusions from patients with well-differentiated ovarian tumors and in 5 of the 11 effusions from patients with poorly differentiated ovarian tumors, whereas the 10 control effusions from patients with various diseases were negative.

Adenocarcinoma, Mucinous↗
PubMed1984

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland.

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.

Antibodies, Monoclonal↗
PubMed1984

Human renal antigen defined by a murine monoclonal antibody.

Fusion of spleen cells from a mouse immunized with a surgical specimen of a human renal carcinoma with murine P3 myeloma cells resulted in the establishment of a hybridoma cell line that secreted a monoclonal antibody (MKi-1), of IgG1 subclass, which preferentially reacted on kidney crude membrane (CM) preparations. This monoclonal antibody was tested by solid-phase radioimmunometric assay and immunofluorescence (IF) on a panel of tumor cell lines and on CM preparations and cell suspensions from surgical specimens of normal and neoplastic tissues. In addition, cryosections of normal and cancer tissues of various histologic types were tested by IF. The expression of the MKi-1 antigen was limited to normal kidney epithelium, renal cancers, some areas in the pancreas, the apical region of some breast ducts, and a proportion (5-50%) of activated lymphocytes. Electron microscopic study by the immunoperoxidase technique on fixed sections from normal kidney showed that MKi-1 stained the brush border of almost all proximal tubules. The molecule recognized by MKi-1 was a single polypeptide chain with a molecular weight of 140,000.

Animals↗
PubMed1983

Immunochemical analysis of the determinant recognized by a monoclonal antibody (MBr1) which specifically binds to human mammary epithelial cells.

A monoclonal antibody (MBr1) raised against a membrane preparation (CM) of a human breast cancer line (MCF-7) and characterized as mammary gland epithelium associated (S. Mènard, E. Tagliabue, S. Canevari, G. Fossati, and M. I. Colnaghi. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res., 43:1295-1300, 1983), was used to biochemically define and partially purify its target antigen. The antigenic activity recognized by MBr1 was unaffected by treatment of MCF-7 cells with trypsin, protease K, or Vibrio cholerae neuraminidase and by heating at 100 degrees but was abolished by treatment with methanol. Since this behavior suggested a glycolipid nature of the MBr1-defined antigen, total lipids were obtained by chloroform:methanol or tetrahydrofuran:phosphate buffer extractions from crude membrane preparations of MCF-7 cells and of breast cancer surgical specimens. Total absorption of MBr1 activity was found by breast cancer lipid extracts, whereas no absorbing capability was detected with a series of highly purified acid and neutral glycolipids or with normal and neuraminidase-treated red blood cells of human, ox, and sheep species. The same pattern of inhibition of MBr1-binding activity was obtained with total lipid extract and both phases after diethyl ether partition. However, when the three extracts were chromatographed on diethylaminoethyl-Sepharose, the antigenic activity was recovered only in the neutral glycolipid fractions. Periodate oxidation of MCF-7 crude membrane preparation abolished MBr1-binding activity, suggesting that the carbohydrate portion of the molecule may constitute the antigenic determinant.

Antibodies, Monoclonal↗
PubMed1983

Generation of monoclonal antibodies reacting with normal and cancer cells of human breast.

From the fusion of the murine myeloma P3- X63-Ag8-U1 with spleen cells from a mouse immunized with the human breast cancer cell line MCF-7, 88 hybridomas producing antibodies reacting with the immunizing cells were obtained. After a first screening on human leukocytes, red blood cells, and platelets and on human and fetal calf serum, three monoclonal antibodies, MBr1, MBr2, and MBr3, with specificity for the immunizing cells were isolated and further characterized. The three monoclonals were tested by isotopic antiglobulin assay and immunofluorescence on a panel of normal cells or cell membrane preparations, including milk epithelial and foam cells; on plasma and milk proteins; on cells or cell membrane preparations from fresh surgical specimens of breast, kidney, and ovarian carcinomas; and on various tumor cell lines. MBr1 and MBr2 had a superimposable reactivity and showed specificity for a structure which seems to characterize both normal and neoplastic mammary gland epithelial cells.

Animals↗
PubMed1979

Antibody binding to membrane of cultured melanoma cells by sera of melanoma patients.

One hundred and nine sera from 75 patients with malignant melanoma and 69 sera from as many healthy donors were assayed by isotopic antiglobulin technique (IAT) on 2 melanoma cell lines. The same picture of reactivity was observed with patients' and healthy donors' sera, and in both groups 35% of the cases were high responders on 1 line and 21% on the other one. The specificity of the reactions was analyzed by absorption experiments using 12 melanoma sera selected for their high binding activity. Pools of human erythrocytes or leukocytes did not remove, except in 1 case respectively, the activity of the sera, suggesting that it was not directed against alloantigens. Quantitative absorption experiments were done with the 2 melanoma lines and with 1 colon carcinoma line. The results, evaluated on the basis of absorption capacity per cell, indicate that the 2 melanoma lines had a similar amount of shared antigens, whereas the colon line was also effective in absorbing out the serum activity, but less frequently and less efficiently. Further experiments performed to analyze the influence of culturing the target cells in presence of fetal bovine serum (FBS), showed that the activity of sera was removed, at various degrees for different sera, by absorption with free FBS, with FBS coupled to Sepharose 4B, and with normal leukocytes cultured overnight with 10% FBS. The same positive melanoma sera became negative when assayed on the same melanoma line cultured in gamma-globulin-depleted human AB serum. In conclusion, in our experimental conditions, the activity of melanoma sera seems mostly directed against components of FBS absorbed on cell membrane during culturing.

Adenocarcinoma↗
PubMed1976

Cellular immune reaction to human malignant melanoma and breast carcinoma cells.

An in vitro microassay was used to study cytotoxic reactivity of lymphocytes of 19 patients with malignant melanoma, 8 patients with breast carcinoma, and 18 normal subjects on cell cultures of malignant melanoma and breast carcinoma. In each of the twelve experiments, peripheral blood lymphocytes from individuals with and without cancer were tested simultaneously on two or three different target cells. Cytotoxic reactivity, evaluated by a comparison of the number of target cells remaining after incubation with lymphocytes with those incubated with medium only, was found in 20 cancer patients (74%) and 13 individuals without cancer (72%). The strength of lymphocyte reactivity of the cancer and of the non-cancer group did not differ significantly. Of the 27 cancer patients, 8 were positive only on the homologous target cells, 7 only on the opposite cells, and 5 on both types; 7 were negative. Short-term melanoma cell cultures were more lysable than were established cell lines; however, no direct correlation between the growth rate during the test period and susceptibility to lysis was seen. The blood group of the lymphocyte donors had no influence on cytotoxic reactivity.

ABO Blood-Group System↗
PubMed1975

Humoral cytotoxicity in melanoma patients and its correlation with the extent and course of the disease.

Complement-dependent cytotoxicity against melanoma cells was demonstrated with a microassay in sera from melanoma patients. The response was tumor-specific and histologic type-specific since 16 out of 52 (30%) melanoma patient sera taken before surgery reacted against melanoma cells, whereas 3 out of 43 (7%) control sera, collected from patients with unrelated tumors and from cancer-free individuals, were positive. The serum activity correlated with the clinical stage of the disease since it was detected in 15 out of 40 patients with stage I and II tumors and in 1 of the 12 patients in stage III. Twelve melanoma patients, clinically tumor-free for to 4 years after surgery, showed no humoral cytotoxicity. A follow-up study of 13 melanoma patients revealed that the cytotoxicity appeared 7-10 days after radical removal of the tumor and that it disappeared if there was no recurrence. The histologic type-specificity was further tested by assaying sera from 16 melanoma and 10 breast-cancer patients simultaneously on both melanoma and breast-cancer cells; a positive reaction was observed in 6 cases of melanoma and in 5 of breast cancer on homologous cells only, in 1 case on the opposite type of cells, and in 3 cases on both types of tumor cells.

Adolescent↗
PubMedDate not supplied

Role of cross-linking agents in determining the biochemical and pharmacokinetic properties of Mgr6-clavin immunotoxins.

Several immunotoxins (ITs) were synthesized by the attachment of clavin, a recombinant toxic protein derived from Aspergillus clavatus, to the monoclonal antibody Mgr6 that recognizes an epitope of the gp185(HER-2) extracellular domain expressed on breast and ovarian carcinoma cells. Conjugation and purification parameters were analyzed in an effort to optimize the antitumor activity and stability of the ITs in vivo. To modulate the in vitro and in vivo properties of the immunotoxins, different coupling procedures were used and both disulfide and thioether linkages were obtained. Unhindered and hindered disulfide with a methyl group linkage ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT) or ethyl S-acetyl 3-mercaptobutyrothioimidate ester hydrochloride (M-AMPT) were obtained by reaction with recombinant clavin, while the monoclonal antibody Mgr6 was derivatized with ethyl 3-[(4-carboxamidophenyl)dithio]propionthioimidate ester hydrochloride (CDPT). To achieve higher hindrance (a disulfide bond with a geminal dimethyl group), Mgr6 was derivatized with the N-hydroxysuccinimidyl 3-methyl-3-(acetylthio)butanoate (SAMBA) and clavin with CDPT. To evaluate the relevance of the disulfide bond in the potency and pharmacokinetic behavior of the ITs, a conjugate consisting of a stable thioether bond was also prepared by derivatizing Mgr6 with the N-hydroxysuccinimidyl ester of iodoacetic acid (SIA) and clavin with AMPT. The immunotoxins were purified and characterized using a single-step chromatographic procedure. Specificity and cytotoxicity were assayed on target and unrelated cell lines. The data indicate that the introduction of a hindered disulfide linkage into ITs has little or no effect on antitumor activity and suggest that disulfide cleavage is essential for activity; indeed, the intracellularly unbreakable thioether linkage produced an inactive IT. Analysis of IT stability in vitro showed that the release of mAb by incubation with glutathione is proportional to the presence of methyl groups and increases exponentially with the increase in steric hindrance. Analysis of the pharmacokinetic behavior of ITs in Balb/c mice given intravenous bolus injections indicated that ITs with higher in vitro stability were eliminated more slowly; i.e., the disulfide bearing a methyl group doubled the beta-phase half-life (from 3.5 to 7.1 h) compared with that of the unhindered, while a geminal dimethyl protection increased the elimination phase to 24 h. The thioether linkage showed its intrinsic stability with a beta-phase half-life of 46 h. The thioether linkage also increased the distribution phase from 17 to 32 min. The in vitro characteristics and in vivo stability of Mgr6-clavin conjugates composed of a methyl and dimethyl steric hindered disulfide suggest clinical usefulness.

Animals↗