Internalization of a monoclonal antibody against human breast cancer by immunoelectron microscopy

Torre, Gabriella Della; Canevari, S.; Orlandi, Rosaria; Colnaghi, Maria I.

doi:10.1038/bjc.1987.72

Cited by 18 publications

(4 citation statements)

References 10 publications

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“…This method (Matzku et al, 1986), is adequate for assessing the persistence of the antigen-antibody complex on the outside of the cell, but does not give information on the further processing of the complex, for which immuno-electron microscopy studies are necessary. A good agreement between the 2 methods (desorption assay and immuno-electron microscopy) was obtained in our laboratory with the MAb MBrl which exhibited partial internalization of its membrane target (Della Torre et al, 1987) and a significant portion (about 35%) of labelled MAb which was non-desorbable by PH 2.8 buffer treatment (data not shown).…”

Section: Discussionmentioning

confidence: 58%

“…Over the past few years we have approached the problem of immunotherapy using immunoconjugates (Canevari et al, 1985;Colnaghi et al, 1987) and, like many others, we had to face a critical factor which affects their efficiency, namely the amount and type of the antigen-antibody complex internalization (Della Torre et al, 1987;Pastan ef al., 1986). As a first step we studied the stability on the cell membrane of the antigen recognized by MOv18 by the PH 2.8 buffer treatment.…”

Section: Discussionmentioning

confidence: 99%

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Human ovarian carcinoma lysis by cytotoxic t cells targeted by bispecific monoclonal antibodies: Analysis of the antibody components

Mezzanzanica

Canevari

Ménard

et al. 1988

Intl Journal of Cancer

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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.

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Section: Discussionmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Human ovarian carcinoma lysis by cytotoxic t cells targeted by bispecific monoclonal antibodies: Analysis of the antibody components

Mezzanzanica

Canevari

Ménard

et al. 1988

Intl Journal of Cancer

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“…The evaluation of the antigen-antibody (Ag-Ab) interactions and the fate of the Ag-Ab complex have already been defined for a series of anti-tumor MAbs, used as single agents (Garnett and Baldwin, 1986;Matzku et al, 1986;Della Torre et al, 1987;Tilgen et al, 1987;Calafat et al, 1988;Yokota et al, 1989), and their relevance in choosing the proper clinical application has been pointed out (Goldmacher et al, 1989;Mezzanzanica et al, 1989;Wargalla and Reisfeld, 1989). In contrast, only a few studies have been devoted to analysis of the binding effect andlor internalization when MAbs were used in combination.…”

mentioning

confidence: 99%

Use of combination of monoclonal antibodies directed against three distinct epitopes of a tumor‐associated antigen: Analysis of cell binding and internalization

Casalini¹,

Mezzanzanica²,

Canevari³

et al. 1991

Intl Journal of Cancer

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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.

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“…Aggregation of gold conjugates can occur due to the repulsive energy forces that separate gold conjugates being lesser than the primary attraction forces (van der Waals forces). This phenomenon will eventually cause the aggregation of gold conjugates [10,15].…”

Section: Temperature and Phmentioning

confidence: 99%

Immunogold Techniques in Electron Microscopy

Murtey¹

2016

Modern Electron Microscopy in Physical and Life Sciences

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Ever since electron microscopy became an important tool in the scientific research, the focus had been mainly on ultrastructural analysis with little success in the development and application of suitable techniques for the localization of macromolecules in cells. The emergence of immunogold techniques in the 1960s managed to fill this gap in serving this function. The aim of this chapter is to equip researchers, postgraduate students, and technicians with essential knowledge to utilize immunogold techniques for ultrastructural investigations in the life sciences. The principles and factors involved have been highlighted to give researchers a quick review of the techniques before embarking on their ultrastructural localization procedures. The advantages and limitations of the four types of immunogold labeling techniques have been discussed.

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Internalization of a monoclonal antibody against human breast cancer by immunoelectron microscopy

Cited by 18 publications

References 10 publications

Human ovarian carcinoma lysis by cytotoxic t cells targeted by bispecific monoclonal antibodies: Analysis of the antibody components

Human ovarian carcinoma lysis by cytotoxic t cells targeted by bispecific monoclonal antibodies: Analysis of the antibody components

Use of combination of monoclonal antibodies directed against three distinct epitopes of a tumor‐associated antigen: Analysis of cell binding and internalization

Immunogold Techniques in Electron Microscopy

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