Document Detail


111In-labeled immunoconjugates (ICs) bispecific for the epidermal growth factor receptor (EGFR) and cyclin-dependent kinase inhibitor, p27Kip1.
MedLine Citation:
PMID:  19409037     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
OBJECTIVE: Probing intranuclear proteins in breast cancer (BC) cells by using radiolabeled antibodies is restricted by delivery barriers presented by cell and nuclear membranes. Our aim was to construct immunoconjugates (ICs) bispecific for epidermal growth factor receptors (EGFRs) and the intranuclear cyclin-dependent kinase inhibitor (CDKI) p27(Kip1) modified with nuclear-localizing sequences (NLSs) to facilitate their nuclear uptake following EGFR-mediated internalization.
METHODS: Bispecific ICs were constructed by first modifying EGF with peptides [GGPKKKRKVGYGCG] harboring NLS from SV-40 large T-antigen (underlined), then conjugating NLS-EGF to anti-p27(Kip1) antibodies through an extended PEO(12)-maleimide linker (Compound 1). Analogous ICs were constructed by using mouse IgG (Compound 2), a disrupted NLS (Compound 3) or omission of the EGF moiety (Compound 4). Binding to EGFR on MDA-MB-468, H2N, or HR2 BC cells and to p27(Kip1) in HELA cell lysate was measured. Internalization and nuclear importation were evaluated. Retention of the ICs in H2N or trastuzumab (Herceptin)-resistant HR2 cells exposed to trastuzumab to modulate p27(Kip1) expression with/without coexposure to the IGF-1 receptor kinase inhibitor, AG1024, was determined.
RESULTS: Trastuzumab (10 microg/mL) unexpectedly decreased p27(Kip1) expression by 1.7-2.4-fold in H2N or HR2 cells. Conjugation of EGF to anti-p27(Kip1) antibodies (Compound 1) decreased the binding affinity of the ICs 7-fold toward EGFR and p27(Kip1). All ICs bound EGFR on MDA-MB-468 cells except Compound 4. Compound 1 was internalized into H2N cells over 48 hours and Compound 2 exhibited 1.6-fold greater nuclear importation in H2N or MDA-MB-468 cells than Compound 3. There was a significantly lower retention of Compound 1 in H2N cells exposed to trastuzumab, compared to unexposed cells, corresponding to decreased p27(Kip1), but in HR2 cells, diminished retention was observed only when these cells were coexposed to trastuzumab and AG]024.
CONCLUSION: We conclude that (111)In-labeled bispecific ICs were constructed that specifically bound EGFR and p27(Kip1). These ICs were internalized into BC cells expressing EGFR and HER2 and imported into the nucleus. Their decreased retention by cells with trastuzumab-modulated p27(Kip1) suggests that they may be useful for probing this CDKI by imaging.
Authors:
Bart Cornelissen; Veerle Kersemans; Kristin McLarty; Lara Tran; Raymond M Reilly
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cancer biotherapy & radiopharmaceuticals     Volume:  24     ISSN:  1557-8852     ISO Abbreviation:  Cancer Biother. Radiopharm.     Publication Date:  2009 Apr 
Date Detail:
Created Date:  2009-05-04     Completed Date:  2009-08-06     Revised Date:  2013-05-08    
Medline Journal Info:
Nlm Unique ID:  9605408     Medline TA:  Cancer Biother Radiopharm     Country:  United States    
Other Details:
Languages:  eng     Pagination:  163-73     Citation Subset:  IM    
Affiliation:
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
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MeSH Terms
Descriptor/Qualifier:
Antibodies, Bispecific / immunology*,  pharmacokinetics
Antibodies, Monoclonal / immunology,  pharmacokinetics,  pharmacology*
Antibodies, Monoclonal, Humanized
Antineoplastic Agents / immunology,  pharmacokinetics,  pharmacology
Breast Neoplasms / immunology*,  metabolism,  therapy
Cell Line, Tumor
Cyclin-Dependent Kinase Inhibitor p27 / biosynthesis,  immunology*
HeLa Cells
Humans
Immunoconjugates / chemistry,  immunology,  pharmacokinetics*
Indium Radioisotopes / pharmacokinetics*,  therapeutic use
Intracellular Signaling Peptides and Proteins / antagonists & inhibitors,  immunology,  metabolism
Receptor, Epidermal Growth Factor / antagonists & inhibitors,  biosynthesis,  immunology*
Chemical
Reg. No./Substance:
0/Antibodies, Bispecific; 0/Antibodies, Monoclonal; 0/Antibodies, Monoclonal, Humanized; 0/Antineoplastic Agents; 0/CDKN1B protein, human; 0/Immunoconjugates; 0/Indium Radioisotopes; 0/Intracellular Signaling Peptides and Proteins; 147604-94-2/Cyclin-Dependent Kinase Inhibitor p27; EC 2.7.10.1/Receptor, Epidermal Growth Factor; P188ANX8CK/trastuzumab

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