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Biomedical subjects

M I Colnaghi

Publications and source records attributed to M I Colnaghi.

At least 145 records · Page 8Linked to original sources
PubMed1989

Re-targeting of human lymphocytes expressing the T-cell receptor gamma/delta to ovarian carcinoma cells by the use of bispecific monoclonal antibodies.

TcR gamma/delta+ lymphocytes represent a small subset homogeneously composed of cytolytic T cells displaying unique motility and homing properties. Since the lytic machinery of these cells can be efficiently triggered by monoclonal antibodies (MAbs) directed to the TcR gamma/delta, such MAbs were used for the construction of bispecific MAbs in conjunction with an MAb specific for ovarian carcinoma cells. Hybrid hybridomas were obtained by fusing the Mov19 MAb (IgG2a)-producing hybridoma with either GI (IgG2a) or A13 (IgG1) hybridomas, secreting MAbs specific for 2 peripheral blood subsets of TcR gamma/delta+ lymphocytes. Hybrid hybridomas producing bispecific MAbs were screened according to their ability to induce ovarian carcinoma (IGROVI) target cell lysis by GI+ or A13+ T cell clones, respectively. The GI-derived GM33.9 bispecific MAb induced selective lysis of Mov19+ ovarian carcinoma target cells only by GI+ clones, whereas the A13-derived AM18.4 MAb was effective only in combination with A13+ clones. Neither the anti-TcR gamma/delta nor the Mov19 parental MAbs (used alone or in combination) induced target-cell lysis. The hybrid nature (IgG1/IgG2a) of the AM18.4 bispecific MAb was indicated by 2-color immunofluorescence experiments. Thus, both ovarian carcinoma and A13+ effector cells were double stained by AM18.4 bispecific MAb followed by PE-conjugated anti-IgG1 and FITC-conjugated anti-IgG2a second reagents. Polyclonal TcR gamma/delta+ cells were obtained by direct stimulation of peripheral blood mononuclear cells with Sepharose bead-conjugated anti-TcR gamma/delta MAbs and IL-2. The lines so obtained contained more than 90% of TcR gamma/delta+ cells after 4 weeks of culture, with an increase in TcR gamma/delta+ cell numbers ranging from 200 to 1,000-fold. These TcR gamma/delta+ cell lines efficiently lysed ovarian carcinoma target cells in the presence of bispecific MAb and may therefore represent a suitable source of effector cells for induction of ovarian carcinoma cell lysis.

Antibodies, Monoclonal↗
PubMed1989

The Aspergillus toxin restriction is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells.

The protein toxin restriction, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of Mr 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restriction toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later. After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites interanalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.

Allergens↗
PubMed1989

Retargeting of T-cell-receptor gamma/delta+ lymphocytes against tumor cells by bispecific monoclonal antibodies. Induction of cytolytic activity and lymphokine production.

We have recently selected MAbs specific for different molecular forms of the TCR gamma/delta (expressed by distinct cell subsets), able to activate TCR gamma/delta+ cells. Two of these reagents (G1 and A13) were used for the construction of bispecific MAbs in conjunction with a MAb (Mov19) directed to ovarian carcinoma cells, using the hybrid hybridoma technique. The G1-derived bispecific MAb GM33.9 efficiently induced lysis of Mov19+ ovarian carcinoma cell lines (IGROVI and SW626) by G1+ clones in a 4-hr 51Cr-release assay. On the other hand, it was ineffective when Mov19- target cells were used. Comparable results were obtained with the A13-derived AM18.4 bispecific MAb when A13+ clones were used as effector cells. Bispecific MAbs were also able to induce secretion of IL-2 and TNF-alpha by TCR gamma/delta+ clones when Mov19+ target cells were present.

Antibodies, Monoclonal↗
PubMed1989

Type 4 chain H expression by bile ductules and hepatocytes in cirrhosis.

The monoclonal antibody MBr1 defines the blood group H determinant with beta 1----3N-acetylgalactosamine linkage (Fuc alpha 1----2Gal beta 1----3GalNAc----R) carried by type 3 or 4 backbone. The distribution of the antigen detected by this antibody was studied immunohistochemically in liver tissues. Although bile ducts with a diameter of more than about 100 microns normally expressed the MBr1-reactive antigen supranuclearly, smaller bile ducts and bile ductules did not express the antigen. In cirrhotic liver, proliferated bile ductules extensively expressed the MBr1-reactive antigen. In spite of the absence in normal liver cells, the antigen was expressed membranously in some cirrhotic liver cells. Under subcellular fractionation, MBr1 reactivity was almost exclusively recovered in the microsomal fraction. By HPTLC immunostaining, the major MBr1-reactive antigen was shown to be carried by type 4 chain H glycolipid (globo-H, Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer). MBr1 reactive glycoprotein was not found. In conclusion, although type 4 chain H glycolipid is not expressed by normal bile ductules and liver cells, it is actively synthesized and expressed by proliferated bile ductules and some of the liver cells in cirrhosis in the absence of any neoplastic change.

Antibodies, Monoclonal↗
PubMed1988

Monoclonal antibodies against doxorubicin.

Hybridomas which secrete monoclonal antibodies (MAbs) reacting with doxorubicin (DXR), a widely used anthracycline cytotoxic antibiotic, have been obtained by fusing NSO/P3 mouse myeloma cells with spleen cells from BALB/c mice immunized with DXR-BSA conjugate. The best producer among the several clones obtained was expanded and the secreted MAb (MAD2) purified and characterized. MAD2 cross-reacts to varying degrees with anthracycline compounds such as some DXR analogues and derivatives, but does not recognize anthracene and anthraquinone structures, with the exception of weakly reacting Mitoxantrone. MAD2 and the panel of MAbs which are at present being purified may become a tool for studying the relevance of different domains of the anthracyclin molecule in terms of biologic activity.

Animals↗
PubMed1988

Activation of mononuclear cells to be used for hybrid monoclonal antibody-induced lysis of human ovarian carcinoma cells.

Recently we reported that cytotoxic T-cell clones can be retargeted to unrelated tumor cells by bispecific monoclonal antibodies (MAbs), anti-CD3 and anti-ovarian carcinoma (alpha OC/TR) (Mezzanzanica et al., 1988). In the perspective of in vivo tumor immunotherapy, as an alternative to cytotoxic T lymphocytes (CTL) from T-cell clones, since human peripheral blood mononuclear cells (PBMCs) without stimulation were quite ineffective, a suitable in vitro activation method was developed to render PBMCs lytic for relevant targets in the presence of the bispecific hybrid MAb alpha OC/TR. This activation protocol was applied to PBMCs from 9 healthy donors (HD) and 6 ovarian carcinoma patients (P) and to tumor-associated lymphocytes (TAL) from 4 ovarian carcinoma P. The method consisted of in vitro stimulation with phytohemagglutinin (PHA) for 2 days, followed by culture with a low dose of recombinant human interleukin-2 (rIL-2) for 6 to 10 days. The antibody-mediated lysis of CTL from HD PBMCs was found to be specifically directed against cells expressing the relevant ovarian tumor antigen when different tumor cell lines and short-term cultures of tumor and normal cells were tested. The antibody-mediated lysis of CTL from P PBMCs or TAL was efficient both on autologous and allogeneic ovarian tumor cells, whereas no reactivity with autologous normal cells was observed and LAK activity was only evident in 1 out of 4 cases. The hybrid antibody induced cytotoxic activity of CTL from P was, however, lower than that of CTL from HD.

Antibodies, Monoclonal↗
PubMed1988

Histopathological characterization of a novel monoclonal antibody, MLuC1, reacting with lung carcinomas.

A monoclonal antibody (MoAb), MLuC1, derived from the fusion of P3-X63-Ag 8-U1 mouse myeloma cells with spleen cells from an HR mouse immunized with the carcinoma cell line SW626, was studied to define its reactivity profile on normal and neoplastic human tissues and its potential clinical applications in lung cancer. Evaluation of paraffin sections using the ABC immunoperoxidase method showed a "pan-epithelial" reactivity; a large majority of epithelial components of organs in the respiratory, digestive and urogenital systems (except liver, rectum and ovary) were immunostained. As regard to neoplastic tissues MLuC1 recognized 84% of lung carcinomas (82% of small cell, 100% of squamous cell, 74% of adenocarcinomas), 86% of breast and 62% of ovarian carcinomas. On the contrary, MLuC1 was non-reactive with the other normal and tumoral non-epithelial tissues. Due to its spectrum of reactivity this MoAb could be useful for different diagnostic purposes such as differential diagnosis and lung cancer cytology.

Adenocarcinoma↗
PubMed1988

Monoclonal antibody detection of carcinoma cells in bone marrow biopsy specimens from breast cancer patients.

The possibility of using immunologic methods for detecting metastatic cells in bone marrow samples from breast cancer patients was investigated. The MBr1 monoclonal antibody, which recognizes a membrane antigen on breast carcinoma cells and is unreactive on normal bone marrow cells, seemed to be an adequate reagent for this kind of approach. When human leukocyte suspensions artificially contaminated with mammary tumor cells were tested by MBr1 immunofluorescence, it was demonstrated that the added tumor cells could be specifically discriminated from normal cells and that as little as one tumor cell in 200,000 could be detected. With the same methodology we screened bone marrow biopsies from breast cancer patients with apparently uninvolved lymph nodes at the moment of surgery. Immunoreactive tumor cells were detected by the MBr1 antibody in 17% of N- patients. None of the bone marrow samples showed any evidence of tumor involvement by conventional histologic analysis.

Antibodies, Monoclonal↗
PubMed1988

Human ovarian carcinoma lysis by cytotoxic T cells targeted by bispecific monoclonal antibodies: analysis of the antibody components.

In the perspective of in vivo therapeutic applications, the monoclonal antibody (MAb) MOv18 was selected for its restricted reactivity with human ovarian carcinoma. Using the pH 2.8 desorption assay, we found that the antigen recognized by MOv18 had a high stability on the cell membrane and poor internalization. Therefore, a therapeutic approach which does not require internalization, i.e., the re-targeting of cytotoxic T lymphocytes (CTL) by bispecific MAbs, was investigated. MOv18 and anti-CD3 MAbs were used to produce bispecific reagents, obtained either by chemical cross-linkage (hetero-conjugates) or by somatic hybridization techniques (hybrid MAbs). The maintenance of the binding reactivity and specificity of the bispecific MAbs was analyzed by solid-phase radioimmunoassay, immunofluorescence and cross-competition tests on the relevant target cells (ovarian carcinoma cell line OVCA 432 for MOv18 and PHA-stimulated peripheral blood mononuclear cells for anti-CD3 MAbs), and on 2 irrelevant tumor cell lines. Bv a 51Cr-release assay the bispecific MAbs were found to efficiently promote, at picomolar concentration, cell lysis by CTL clones, but the specificity pattern was wider than that predicted by the binding studies. The F(ab')2 fragment of one hybrid MAb mediated a lysis which was just as efficient as the entire MAb on the relevant target cells and allowed specific lysis to be distinguished from Fc-receptor-mediated lysis. Human immunoglobulins were unable to compete with the Fc receptor binding of the hybrid MAbs and therefore, in the perspective of in vivo applications, Fc fragment removal seems to be an essential step. Analysis of the bispecific reagents indicated that hybrid MAbs are superior to the heteroconjugate as far as storage stability is concerned.

Animals↗
PubMed1988

Anti-ovarian carcinoma anti-T3 heteroconjugates or hybrid antibodies induce tumor cell lysis by cytotoxic T-cells.

In the perspective of therapeutic in vivo targeting for T-cell attack, the monoclonal antibody (MAb) MOv18, selected for its restricted reactivity with human ovarian carcinoma, and an anti-T3 MAb were used to produce heteroconjugate or hybrid antibodies derived by fusion of relevant hybridomas. Specificity and activity of bispecific MAbs were analyzed by solid-phase RIA, immunofluorescence and a 51Cr-release assay on the ovarian carcinoma cell line OVCA 432, which expresses the relevant tumor-associated antigen, and on several irrelevant tumor cell lines. Both reagents efficiently promoted, at picomolar concentration, target cell lysis by cytotoxic T-cell (CTL) clones. Although the pattern of tumor cell lines which were lysed was wider than that predicted by binding studies, further studies using a double-determinant immunoradiometric assay confirmed the specificity of MAb targeting. Analysis of reagents indicated that the hybrid MAb was superior to the heteroaggregate as far as purification recovery and storage stability were concerned. Besides CTL clones, peripheral blood lymphocytes could also be used as cytolytic effectors, provided that a suitable in vitro activation scheme was used.

Antibodies, Heterophile↗
PubMed1988

Immunoconjugate generation between the ribosome inactivating protein restrictocin and an anti-human breast carcinoma MAB.

In the perspective of therapeutic approaches the monoclonal antibody, MBrl, with a quite restricted spectrum of reactivity for human breast carcinoma, was coupled to restrictocin (Res), a ribosome inactivating protein produced by Aspergillus restrictus. In a cell-free system this toxin was found to have an activity comparable to that of other plant toxins, but its in vitro toxicity was shown to be low on different cell lines. Three batches of MBr1-Res conjugate were prepared and their specificity, efficiency, and maximum level of cytotoxicity were analyzed on the cell line MCF-7 expressing the relevant antigen, on several irrelevant tumor cell lines, and on normal cells. Conjugates were from 600 to 1500 times more efficient than the uncoupled derivatized Res towards MCF-7 cells and were completely ineffective on the other target cells. The antigen-driven cytotoxicity was confirmed by the nontoxicity of an irrelevant conjugate on MCF-7 cells. The cytotoxic efficiency of MBr1-Res was low when compared to the binding level of MBr1 at the same concentration and a portion of treated cells (from 10% to 30%) survived the treatment. The heterogeneity of expression of the relevant antigen, together with its only partial internalization, could account for these limitations. The lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin were tested as potentiating agents but in both cases the cytotoxicity remained unmodified. A neutralization assay performed on a xenogenic model indicated that the MBr1-Res conjugate was capable of reducing the tumor take. These data indicate the possibility of using the Res to prepare a reproducible and highly selective breast cancer conjugate. However, there are still a number of problems which must first be solved before we can consider its clinical application.

Allergens↗
PubMed1988

The antitumor monoclonal antibody MOv2 recognizes the Lewis A hapten.

Monoclonal antibody MOv2, produced against ovarian carcinoma, was previously found to bind a carbohydrate epitope (CAMOv2) present on mucins, glycoproteins and a neutral glycolipid. In this paper, the structure of the carbohydrate epitope is determined by immunological reactivity with purified glycolipids and oligosaccharides. Using solid-phase radioimmunoassay and immunostaining of thin layer chromatograms, MOv2 binds strongly to Le(a)-active pentasaccharide ceramide. A smaller neutral glycolipid also weakly binds MOv2. Fifty percent inhibition of binding to Le(a)-active pentasaccharide ceramide is achieved with approximately 8 microM concentration of lacto-N-fucopentaose II (LNF II). Lacto-N-tetraose (LNT) also partially inhibits at about 10(3) times higher concentration suggesting that the faster migrating glycolipid antigen contains this carbohydrate sequence. Binding to Le(a)-active hapten is further confirmed by the specific inhibition of binding by authentic anti-Le(a) monoclonal antibodies but not by anti-Le(b) MOv2 antibody in a serum assay among healthy blood donors also supports these results. In conclusion, we have obtained direct evidence from several independent experiments that antibody MOv2 recognizes the Le(a)-active hapten.

Antibodies, Monoclonal↗
PubMed1988

Association of monoclonal antibody-recognized antigen with steroid receptors in gynecologic tumors.

In an attempt to differentiate between the biochemical characteristics of hormonal-dependent and independent gynecologic tumors, monoclonal antibodies (mABs) MBr1 and MBr3 were used to detect tumor antigens. Steroid receptors and MBr1 and MBr3 determinants were assayed independently in a double-blinded manner. Of the 45 gynecologic tumors analyzed, 16 of 20 (80%) with both progesterone receptors (PR) (greater than 10 fm/mg protein) and estrogen receptors (ER) (greater than 5 fm/mg protein) also contained high levels of MBr1 determinant. However, these receptors were not quantitatively related to MBr1 activity. The 15 of 17 (88%) tumors that contained receptors below the discriminant levels also had low or undetectable levels of MBr1 activity. In tumors where only either PR or ER was present, 2 of 2 PR+/ER- tumors and 2 of 6 PR-/ER+ tumors expressed MBr1 antigen. No relationship of MBr3 antigenic activity to the receptors was established. These results show that MBr1-recognized antigen is expressed largely in steroid receptor-positive tumors and these data suggest that either the antigen itself or its carrier protein may be regulated by estrogen. Like steroid receptors, MBr1 antigens may have important prognostic value in gynecologic tumors.

Antibodies, Monoclonal↗
PubMed1987

The effect of human serum on the binding activity of radiolabelled monoclonal antibodies.

Three murine monoclonal antibodies (MoAbs), MBr1 and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabelled MoAbs were analyzed for their "in vitro" stability in human blood. They were incubated at 37 degrees C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

Animals↗
PubMed1987

Immunochemical characterization and radioimmunometric detection of molecules shed by human ovarian cancer.

Monoclonal antibodies (MAbs) OC125, MOv2 and MOv8 recognize the CA125, CAMOv2 and CAMOv8 epitopes, respectively, which are associated with human ovarian carcinomas and are shed by these tumors. The biochemical analysis of the recognized antigens and epitopes has been performed to investigate the possibility of exploiting their expression, if any, on the same molecules. Cross-competition experiments clearly indicated that the 3 epitopes are distinct. Biochemical analysis was performed on a cyst fluid from an ovarian cystadenoma. Heat treatment and periodate oxidation indicated that CAMOv2 and CAMOv8 are saccharidic and suggested that CA125 is a conformational epitope to which both saccharides and the protein backbone could contribute. By gel-filtration chromatography CA125 activity was eluted into 2 peaks of high molecular weight, whereas CAMOv2 and CAMOv8 activities were associated with a single broad peak which included CA125-positive fractions. Isopycnic centrifugation showed that CA125, CAMOv2 and CAMOv8 carrying molecules had the same high density (1.46 g/ml) in the first peak, whereas CA125 molecules had a lower density (1.41 g/ml) in the second peak. Double-determinant immunoradiometric assays, carried out using different combinations of the MAbs, indicated that in the cyst fluid molecules expressing both CAMOv2 and CAMOv8 could be detected, whereas CA125 was carried by different molecules. CAMOv2 and CA125 could however be expressed on the same molecule in 2 out of 9 ascitic fluids from ovarian carcinoma patients. Taken together, these data indicate that CA125 and CAMOv2-CAMOv8 were only occasionally expressed on the same molecules. Therefore, the use of the CA125-CAMOv2 combination could not increase the sensitivity achievable by using each respective simultaneous immunoradiometric assay.

Antigens, Neoplasm↗