Enhanced Binding Activity Observed Between Anti-Carcinoembryonic Monoclonal Antibodies

Wellerson, Ralph; Kaplan, Paul

doi:10.1089/hyb.1986.5.199

Cited by 6 publications

(4 citation statements)

References 8 publications

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“…The central feature of this assay first takes advantage of the fact that a bivalent IgG molecule can bind in cyclic manner to a multivalent antigen (63,64), producing a stable complex containing one or two IgG molecules per p53 tetramer (59). Previous biochemical studies have also indicated that the number of antibodies bound to one tetrameric p53-DNA complex can be quantitated by counting the integral number of stable, intermediate IgGp53-DNA complexes separated by native gel electrophoresis (59).…”

Section: The Development Of An Immunochemical Assay To Quantitate In mentioning

confidence: 99%

Stoichiometric Phosphorylation of Human p53 at Ser315Stimulates p53-dependent Transcription

Blaydes¹,

Luciani²,

Pospı́šilová³

et al. 2001

Journal of Biological Chemistry

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p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser 315 phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser 315 can be detected in vivo, whether steady-state Ser 315 phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser 315 where one, two, three, or four phosphates/ tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser 315 phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser 315 site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser 315 after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

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Section: The Development Of An Immunochemical Assay To Quantitate In mentioning

confidence: 99%

Stoichiometric Phosphorylation of Human p53 at Ser315Stimulates p53-dependent Transcription

Blaydes¹,

Luciani²,

Pospı́šilová³

et al. 2001

Journal of Biological Chemistry

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“…Polyclonal antisera produced by rabbits immunized with protein conjugates of either l -thyroxine or 3,5,3‘-triiodo- l -thyronine were reported to bind the corresponding soluble antigens with a high degree of positive cooperativity. In contrast to the scarcity of literature on allosteric effects due to antigen binding-dependent conformation changes in the antibody, many laboratories have reported that protein antigens can display positive cooperativity in the binding of multiple antibodies to different epitopes on the antigen ( − ). In each of these latter examples, however, the positive cooperativity was thought to arise either from conformation changes in the protein antigen that occurred as a consequence of antibody binding ( − ), or from cyclic structures that could form as a consequence of the multivalency of both the antibody and the protein antigen ( − ).…”

mentioning

confidence: 99%

“…In contrast to the scarcity of literature on allosteric effects due to antigen binding-dependent conformation changes in the antibody, many laboratories have reported that protein antigens can display positive cooperativity in the binding of multiple antibodies to different epitopes on the antigen ( − ). In each of these latter examples, however, the positive cooperativity was thought to arise either from conformation changes in the protein antigen that occurred as a consequence of antibody binding ( − ), or from cyclic structures that could form as a consequence of the multivalency of both the antibody and the protein antigen ( − ). In either case, binding-induced conformation changes in the antibodies were neither postulated nor required to rationalize the experimental observations.…”

mentioning

confidence: 99%

Allosteric Binding Properties of a Monoclonal Antibody and Its Fab Fragment

et al. 2002

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Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.

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“…In contrast, studies using two MAbs with high individual in vivo localization indices for xenografts of colon carcinoma (in nude mice) gave significantly enhanced labelling indices when both were injected together (Munz et al, 1986). MAbs against certain epitopes on carcinoembryonic antigen (CEA) were also shown to increase binding of MAb to other epitopes (Wellerson and Kaplan, 1986). It is evident from these studies that the use of MAb cocktails needs to be assessed for individual tumours but that the presence of two MAbs with high individual labelling indices may be superior to either alone.…”

Section: Mabs Against Tumour-associated Antigens On Melanoma Cells-anmentioning

confidence: 99%

Preclinical and Phase I Studies of Monoclonal Antibodies in Melanoma: Application to Boron Neutron Capture Therapy of Melanoma

Hersey

1989

Pigment Cell Research

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Monoclonal antibodies (MAbs) provide an attractive method of selectively localizing sufficient boron atoms around tumour cells to capture neutrons. Assuming that 10(8)-10(10) 10B atoms are needed for one capture event and that 10(3)-10(4) atoms can be coupled to each antibody molecule, then 10(5)-10(6) antibody molecules gathered on an individual cell will destroy that cell. Binding to normal tissues, on the other hand, would need to be at least 20-fold less than that to tumour tissues to avoid toxic effects of neutrons on surrounding tissues. Preclinical studies in animals show that several MAbs may bind to melanoma cells in sufficient quantities in vitro to localize the required amount of Boron per cell. Whether this will occur in vivo, however, may depend not only on antigen density but a variety of other properties of the tumour cells and MAbs. These include the Ig class and affinity of the antibody and whether the antibody is internalized into the tumour cell. The ratio of uptake between tumour and normal tissue is governed by such factors as the percentage of tumour cells within a tumour expressing the antigen and whether the MAb react with normal tissues. Use of Fab or F(ab)2 preparations of the MAb may increase the uptake ratio by preventing uptake of MAb by cells with Fc receptors. In contrast to preclinical animal studies, tumour/normal tissue uptake ratios in phase I studies in humans have been disappointingly low.(ABSTRACT TRUNCATED AT 250 WORDS)

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Enhanced Binding Activity Observed Between Anti-Carcinoembryonic Monoclonal Antibodies

Cited by 6 publications

References 8 publications

Stoichiometric Phosphorylation of Human p53 at Ser315Stimulates p53-dependent Transcription

Stoichiometric Phosphorylation of Human p53 at Ser315Stimulates p53-dependent Transcription

Allosteric Binding Properties of a Monoclonal Antibody and Its Fab Fragment

Preclinical and Phase I Studies of Monoclonal Antibodies in Melanoma: Application to Boron Neutron Capture Therapy of Melanoma

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