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Biomedical subjects

M I Colnaghi

Publications and source records attributed to M I Colnaghi.

At least 109 records · Page 6Linked to original sources
PubMed1992

Characterization of a mouse-human chimeric antibody to a cancer-associated antigen.

In an attempt to obtain a therapeutic antibody, the murine monoclonal antibody (MAb) MBrI (IgM,k), directed against human carcinomas, was converted in a mouse/human chimeric MAb of gamma I isotype. The chimeric MAb, gamma I CHI-MBrI, retains the ability to specifically bind tumor cells and tissues with no modification in its binding to the normal material tested. gamma I CHI-MBrI recognizes mucins and high-molecular-weight glycoproteins carrying the antigenic determinant and stains a neutral glycolipid extracted from MCF-7 cells. The chimeric and the murine MBrI efficiently cross-inhibit each other on the reference cell line MCF-7 and the calculated affinity constants amount to 3.8 x 10(7) and 1.7 x 10(8) M-1, respectively. The human constant region allows gamma I CHI-MBrI to bind with the FcR on the human monocytic cell line U937 and to efficiently mediate antibody-dependent cellular cytotoxicity in the presence of human lymphocytes activated by IL2. In addition, gamma I CHI-MBrI, like the murine MBrI, mediates complement-dependent tumor-cell lysis. Thus, by modelling a molecule with reduced size and increased functional characteristics, we have obtained a reagent which is more suitable for in vivo therapeutic approaches.

Animals↗
PubMed1992

Membrane association and shedding of the GPI-anchored Ca-MOv18 antigen in human ovary carcinoma cells.

The antigen recognized by the MOv18 MAb (Ca-MOv18) was recently shown to be a glycosylphosphatidylinositol (GPI)-linked protein. In this report we show that GPI-anchorage is not limited to IGROVI cells nor to other ovary carcinoma cell lines, but Ca-MOv18 was also found to be sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment on fresh ovarian cancer cells. Furthermore, we found a heterogeneous sensitivity of Ca-MOv18 to PI-PLC cleavage, not only among the different cells studied but also in different experiments performed on the same cell line, during extended periods of time in culture. Sensitivity to PI-PLC cleavage was determined by immunofluorescence on live cells and by double-determinant radioimmunoassay of the antigen released in the supernatant. The specificity of the PI-PLC cleavage was demonstrated as follows: (a) TX114 solubilized Ca-MOv18 shifts from the detergent to the aqueous phase after treatment with PI-PLC; (b) on membrane preparations, PI-PLC specifically released a fraction of the antigen, which is distinct from the weakly associated form released by high-salt treatment; (c) Ca-MOv18 from IGROVI expressed the cross-reacting determinant (CRD), which is characteristic of GPI-linked molecules. The absence of CRD expression on the spontaneously released protein and the possibility of artificially inducing antigen shedding during the resynthesis of Ca-MOv18 which follows bacterial PI-PLC treatment are interesting points which need to be further investigated in order to understand the physiology of the Ca-MOv18 tumor antigen.

Antibodies, Monoclonal↗
PubMed1992

Glycolipids carrying Le(y) are preferentially expressed on small-cell lung cancer cells as detected by the monoclonal antibody MLuC1.

The monoclonal antibody MLuC1, which reacts strongly with a high percentage of small-cell lung cancers (SCLC), as well as with various human carcinomas, has been used to immunochemically characterize the recognized epitope (CaMLuC1). To this aim 3 different approaches were adopted: (1) immunoblotting/immunostaining of extracts from various tumor-cell lines; (2) inhibition of binding by purified oligosaccharides; (3) direct binding to oligosaccharide-protein conjugates. All of these experiments indicate that CaMLuC1 is present on the Le(y) blood-group structure heterogeneously expressed on various glycoproteins and glycolipids. The expression of the glycoconjugates carrying Le(y) was then analyzed on breast and lung cancers and on their normal counterparts. Our overall results suggest that SCLC produce Le(y)-active glycolipids in higher amounts compared to other tumors of the same or of a different oncotype, as well as normal lung cells, thus indicating an SCLC-specific modification of the glycosylation pathways.

Antibodies, Monoclonal↗
PubMed1992

Production and characterization of two monoclonal antibodies directed against the integrin beta 1 chain.

The production and characterization of two new monoclonal antibodies (MAbs), designated MAR4 and MAR5, raised against the partially purified alpha 5 beta 1 integrin, are described. The reactivity of these 2 MAbs on tumor cell lines indicated that they reacted on all the cells expressing the beta 1 subunit independently of the alpha 5 expression. Both MAbs were found to immunoprecipitate on 3 cell lines, a protein of 120 KD corresponding to the molecular weight be the beta 1 chain, in addition to proteins of other MW corresponding to the alpha subunits differentially expressed by these cells. The cross-competition experiments showed that MAR4 and MAR5 recognize the same epitope. These 2 MAbs seem to be useful reagents for the characterization of the VLA expression in tumor cells.

Animals↗
PubMed1992

An anti-doxorubicin monoclonal antibody modulates kinetic and dynamic characteristics of the drug.

A monoclonal antibody (MAb) directed against doxorubicin (DXR), denominated MAD II, was found to exert an antidotal action by modulating the kinetic and dynamic characteristics of the drug. In vitro, MAD II has been found to reduce the cytotoxicity of DXR and the drug uptake on spleen lymphocytes more efficiently than on tumor cells (P388 leukemia cells). In vivo, the anti-DXR MAb modified the drug distribution; the drug uptake was found to be reduced in the intestine and myocardial tissues and increased in the tumor, liver and spleen. In mice treated with DXR, the administration of anti-DXR MAb exerted an antidotal activity which was proved by the reduction in body-weight loss and mortality. In contrast, the therapeutic efficacy of the drug in P388-tumor-bearing mice was not influenced by the anti-DXR MAb.

Animals↗
PubMed1992

Characterization of two monoclonal antibodies directed against the 67 kDa high affinity laminin receptor and application for the study of breast carcinoma progression.

Two monoclonal antibodies (mAbs), designated MLuC5 and MLuC6, were produced against a human small cell lung carcinoma cell line. They were found to exhibit a superimposable reactivity on different cell lines and on platelets. Moreover, they both immunoprecipitated a 67 kDa molecule from the membrane of the reference target cells. Immunodepletion and cross-inhibition tests indicated that the two mAbs recognize two epitopes closely localized on the same molecule. The MLuC5 mAb was further characterized for its reactivity on platelets. Immunoprecipitation and ELISA assays demonstrate that this mAb recognizes the 67 kDa high affinity laminin receptor. MLuC5 reactivity was evaluated by immunohistochemistry on a variety of normal and tumor tissues, in particular breast specimens including normal epithelium, dysplastic lesions, in situ carcinomas, invasive primary carcinomas and distant metastases. The laminin receptor was found to be strongly expressed in 50% of the infiltrating carcinomas, whereas in situ carcinomas and benign lesions, as well as the normal mammary epithelium, were only weakly and focally positive. In metastatic lesions MLuC5 reactivity was only found in 11% of the samples tested, independently of the site of origin of the lesion.

Animals↗
PubMed1992

Bispecific antibodies and retargeted cellular cytotoxicity: novel approaches to cancer therapy.

We have used a relatively new technology to increase the number of human lymphocytes that will react with human ovarian carcinoma cells. This technology, often called "retargeting of the immune system," can temporarily redirect the activity of immune cells that were originally committed to react with foreign substances other than cancer cells. In the example presented here, the antitumor effects of retargeted human T lymphocytes, collected from normal donors, were tested in immunodeficient mice with a human ovarian carcinoma line growing intraperitoneally. We retargeted T cells in vitro with a bispecific antibody that reacted with the T cell receptor complex and with a cell-surface antigen expressed by the ovarian carcinoma cells. Retargeted lymphocytes, injected intraperitoneally into mice 4 days after intraperitoneal injection of the tumor cells, impeded tumor growth and doubled the host survival time. These findings provide support for the concept that treatment of ovarian cancer patients with retargeted T cells could prove beneficial.

Animals↗
PubMed1992

p185 HER2/neu epitope mapping with murine monoclonal antibodies.

In order to obtain further information on the biological role of the HER2/neu oncoprotein, 7 new monoclonal antibodies (MAbs) were produced against the p185HER2 extracellular domain. These MAbs, together with two others previously produced, were used to investigate the p185HER2 expression in breast carcinomas and compare the recognized antigenic determinants. The 7 reagents (MGR4,5,6,7,8,9 and 10), were shown to define five distinct epitopes. Three of these MAbs (MGR5,7,10), as well as one previously produced (MGR2), recognize the same epitope (Epitope-1) which seems, therefore, to be highly immunogenic for the murine immune system. Epitope-2 recognized by the MGR4 MAb, appears to be closely related to epitope-1 due to a cross inhibition between MGR4 and MGR10, but not MGR2. Epitope-2 is the only one of the 5 also present on the product of the neu oncogene, the rat analogue of the human HER2/neu gene. None of the reagents against epitope-1 and epitope-2 were found to mediate receptor internalization, whereas MGR6 as well as a previously produced MAb (MGR3), both of which define epitope-3 and MGR8 which defines epitope-4, were found to do so. Epitope-4 like the neu-specific peptide recognized by the reference c-neu Ab3 MAb, was detectable on all p185HER2 positive breast cancer, independently from the quantitative content of the oncoprotein, at variance with the other 4 epitopes whose availability on p185HER2 for the relevant MAbs varied with the degree of overexpression. Epitope-5, recognized by the MGR9 MAb, on the contrary to the other epitopes, was prevalently localized at the basal membrane level of the tumor nodule.

Animals↗
PubMed1992

Production and characterization of a monoclonal antibody against the ribosome inactivating protein alpha sarcin.

In order to improve the purification of immunoconjugates containing alpha sarcin, a ribosome-inactivating protein, and in the attempt to define the enzymic region of the toxin, MAbs against alpha sarcin were produced. From 5 fusions, by adopting a short period of immunization and very low doses of the immunogen, 10 anti-toxin-producing clones were obtained. One of them, named MAsg2 (IgG2b), due to its specific reactivity and secreting properties, was selected for further characterization. MAsg2 was found to recognize an epitope which is common to two, i.e. alpha sarcin and clavatin, of the three different aspergillins tested, but is not involved in the active site of the toxins.

Animals↗
PubMed1992

Biochemical characterization of Trop-2, a cell surface molecule expressed by human carcinomas: formal proof that the monoclonal antibodies T16 and MOv-16 recognize Trop-2.

Trop-2 is a cell surface structure recognized by the 162-46.2 mAb and expressed by most human carcinomas. Since the 162-46.2 mAb works poorly in immunoprecipitation, to characterize the structure of Trop-2 we searched for other mAbs directed against this molecule. Selection of candidates was performed by analyzing the characteristics of mAbs directed against epithelial cells and by comparing the staining pattern of each mAb with the one of the 162-46.2 on frozen sections of human epidermis. Two mAbs, T16 and MOv-16, were selected for further analysis. Formal proof that candidate mAbs reacted with Trop-2 was obtained by comparing their binding patterns to mouse L cells transfected with the Trop-2 gene by genomic DNA transfection and selected by FACS using the FITC-162-46.2 mAb. In immunofluorescence FACS analysis the FITC-T16 and FITC-MOv-16 mAbs specifically stained Trop-2 transfectants. The specificity of binding was confirmed by selective blocking of the staining by the respective unconjugated mAb. Interestingly, cross-blocking studies indicated that the 162-46.2, T16 and MOv-16 mAbs recognize the same epitope or closely spaced ones on the Trop-2 molecule. T16 and MOv-16 efficiently immunoprecipitate Trop-2 from Trop-2 transfectants and from the human cell line OVCA-432, indicating that it is a cell surface glycoprotein, with an apparent molecular weight of 57 kD in non-reducing conditions. A weaker band of 38 kD is often co-precipitated with the 57 kD form in an apparently specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Targeting of T or NK lymphocytes against tumor cells by bispecific monoclonal antibodies: role of different triggering molecules.

MAbs directed against triggering surface molecules expressed by T lymphocytes (CD3, TCR, CD2, CD28) or by NK cells (CD2, CD16) are able to induce the functional program of these cells. These MAbs represent suitable reagents to construct biMAbs directed against TAA, in order to specifically target effector lymphocytes against tumor cells. Anti-CD3/anti-EGF-R biMAbs were constructed to specifically direct T lymphocytes against EGF-R+ tumor cells. Such biMAb are able to induce cytolysis of EGF-R+ tumor cell lines (A431, IGROV, KATO-III and U-87) by cytolytic CD3+ effector lymphocytes while tumor cells having low or absent expression of EGF-R were not lysed. In addition, both cytolytic T (CD8+) cells and non-cytolytic (CD4+) IL-2-expanded lymphocytes were able to secrete lymphokines upon contact with EGF-R+ tumor cells. To target NK cells against NK resistant ovarian carcinomas, we used an anti-CD16 Mab (IgG1) together with an anti-ovarian carcinoma MAb (IgG2a), to construct biMAbs using the hybrid hybridoma technique. The hybrid IgG1/IgG2a biMAb triggered the specific lysis of relevant target cells by resting NK cells and by a subset of NK clones. In addition, some TCR gamma/delta+ clones but not TCR alpha/beta+ clones could be targeted by the biMAb.

Antibodies, Monoclonal↗
PubMed1992

Possible targets on carcinoma for bMAb retargeting of lymphocyte or drug cytotoxicity.

Retargeting of drugs or lymphocyte cytotoxicity through bi-specific monoclonal antibodies (bMAbs) has been proven a therapeutic tool against human carcinoma both in pre-clinical in vitro and in vivo studies. Some of these reagents have already been introduced into clinical trials and preliminary results appear to be promising. However, improvement of the specificity of this approach could be achieved by selecting more suitable target molecules on tumor cells. The research focused on developing MAbs directed against molecules with tumor-restricted distribution and homogeneous expression. Cell-membrane receptors for nutrients or growth factors which operationally represent tumor-specific molecules due to their overexpression, could be considered appropriate targets. Several bMAb anti-nutrients (the folate-binding protein) or growth-factor receptor (c-erbB1, c-erbB2)/anti-triggering molecules have been generated and all were able to efficiently retarget the relevant population of lymphocytes on tumor cells. In order to design a more reliable and selective therapeutic tool, the following parameters were analyzed: correlation between cytotoxicity and antigen level, potency and possible modulation of the target molecule.

Antibodies, Monoclonal↗
PubMed1991

Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein.

Monoclonal antibodies MOv18 and MOv19, raised against a membrane preparation of an ovarian carcinoma surgical specimen, react with a surface antigen present on the majority of nonmucinous ovarian malignant tumors tested but not with normal adult tissue (S. Miotti, S. Canevari, S. Ménard, D. Mezzanzanica, G. Porro, S. M. Pupa, M. Regazzoni, E. Tagliabue, and M. I. Colnaghi, Int. J. Cancer, 39: 297-303, 1987). This surface antigen was purified as a soluble glycoprotein (molecular mass, 36-38 kDa) released from the cell surface of an ovarian carcinoma cell line (IGROV1) by digestion with Bacillus thuringiensis phospholipase C. Immunoblotting demonstrated that the purified protein reacted with MOv18 and MOv19 and that treatment of the purified preparation with N-glycanase resulted in a protein with a molecular mass of 27 kDa. The NH3-terminal amino acid sequence of the purified antigen was determined. This sequence is highly homologous to an internal stretch of 27 amino acids located near the NH3 terminus of human folate-binding protein. An oligonucleotide probe was synthesized and used to screen an IGROV1 ovarian carcinoma, lambda gt11 complementary DNA library to obtain three complementary DNA clones. The complete nucleotide sequence of one of these complementary DNA clones was determined. This sequence is nearly identical to that of a folate-binding protein clone obtained from the Caco-2 human carcinoma cell line. In addition, the nucleotide sequence of the 5'-untranslated region of the other two clones was determined. This region of all three clones was different. The product of the Caco-2 folate-binding protein clone expressed in Chinese hamster ovary cells was recognized by the MOv18 and MOv19 antibodies, confirming that the antigen and folate-binding protein are one and the same. Furthermore, a cell line that binds the MOv18 and MOv19 antibodies expressed increased levels of folate-binding protein mRNA compared with a cell line that does not bind these antibodies. These results indicate that the MOv18 and MOv19 monoclonal antibodies bind to at least one form of folate-binding protein and that this protein, which is evidently overexpressed in certain malignant tumors, may provide a suitable target for immunotherapy with these antibodies.

Amino Acid Sequence↗
PubMed1991

Prognostic significance of HER-2/neu expression in breast cancer and its relationship to other prognostic factors.

Archival surgical specimens from 1,210 female breast cancer patients treated between 1968 and 1971 and with a 19-year follow-up were reanalyzed with special reference to several parameters, such as size of the primary tumor, axillary nodal involvement, histologic grade, degree of inflammatory infiltrate (LPI) of the tumor and expression of the neu oncoprotein (p185) as detected by immunohistochemistry. In a multifactorial analysis the 4 former factors were found to be independent prognostic parameters. Over-expression of p185 was found to be related to tumor size and grade and to LPI but not to pathologic nodal status. Over-expression of p185 showed a negative impact upon survival in node-positive but not in node-negative patients. However, in the subset of node-negative patients without LPI, p185 over-expression showed the same correlation with a poor prognosis as in node-positive patients. In contrast, in node-negative and LPI-positive patients, p185 over-expression correlated with a good prognosis. Also, the prognosis of patients with positive nodes, presence of LPI and no p185 over-expression was similar to that of patients with negative nodes, absence of LPI and p185 over-expression.

Age Factors↗
PubMed1991

Bispecific monoclonal antibodies directed to CD16 and to a tumor-associated antigen induce target-cell lysis by resting NK cells and by a subset of NK clones.

CD16 surface antigens represent activatory molecules in CD3-16+ NK cells. In order to target NK cells against relatively NK-resistant ovarian carcinomas, we used an anti-CD16 monoclonal antibody (MAb) (VD4), together with an anti-ovarian carcinoma-associated antigen (MOV19), to construct biMAbs. To this end, hybrid hybridomas were generated by fusing a TK-deficient VD4 hybridoma mutant with a HGPRT-deficient MOV19 hybrid. Supernatants from hybrid hybridomas that had been selected in HAT medium were screened for their ability to induce a CD3-16+ NK clone to lyse an MOV19+ ovarian carcinoma cell line in a 4-hr 51Cr-release assay. The NMB.45 hybrid hybridoma secreted a biMAb which triggered lysis of MOV19+ but not of MOV19- target cells. Some degree of target cell lysis was also observed with MOV19 MAb (due to ADCC mechanisms), while the VD4 MAb was ineffective. HPLC fractionation of MAbs secreted by the hybrid hybridoma made it possible to identify 4 different peaks, one of which appeared to contain functional biMAb molecules. HPLC-purified biMAb (100 ng/ml) induced resting PBL to lyse the "NK-resistant" IGROVI ovarian carcinoma cell line. Fresh MOV19+ tumor cells were also lysed, although with lower efficiency. When IL-2-activated lymphocytes were used as a source of effectors, biMAb caused only minor increases in the IL-2-induced cytolytic activity. Further analyses of the effect of biMAb were performed at the clonal level. Among CD3-16+ NK cell clones, a clear enhancing effect could be observed only in GL183+ but not in GL183- clones. In CD3+ cytotoxic clones a triggering effect could be detected in one out of 4 TCR gamma/delta+ clones but not in TCR alpha/beta+ clones.

Antibodies, Monoclonal↗
PubMed1991

Use of combination of monoclonal antibodies directed against three distinct epitopes of a tumor-associated antigen: analysis of cell binding and internalization.

Three monoclonal antibodies (MAbs), MOv17, MOv18 and MOv19 with tumor-restricted specificity for human ovarian carcinoma, were tested alone or in double combination with the aim of analyzing their binding and internalization behavior on different in vitro cell lines. Biochemical studies indicated that the 3 MAbs were directed against 3 epitopes of the same 38 kDa surface molecule. By immuno-electron-microscopy they exhibited a different internalization behavior since MOv17 induced evident endocytosis through coated vesicles, whereas MOv18 gave rise to occasional uncoated vesicles and MOv19 was completely unable to promote internalization of the relevant molecule. When tested 2 by 2 there was a binding synergy in one of the 9 possible combinations (125I-labelled MOv18 and unlabelled MOv19), but no change in the internalization behavior. The binding synergy, which was highly reproducible, was temperature-dependent and was also evident on glutaraldehyde-fixed cells. A metabolism involvement is therefore unlikely. This could be attributed to an easier accessibility of the CaMOv18 due to a conformational change of the molecule after MOv19 MAb binding.

Allosteric Regulation↗