Selection of tumor-specific internalizing human antibodies from phage libraries

Poul, Marie‐Alix; Becerril, Baltazar; Nielsen, Ulrik B.; Morisson, Peter; Marks, James D.

doi:10.1006/jmbi.2000.4026

Cited by 184 publications

(176 citation statements)

References 47 publications

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“…The protocols of Poul et al 6 were followed with slight modifications. Cells were plated and grown to about 80% confluency on Lab -Tek chamber slides (eight wells; Nunc, Naperville, IL ).…”

Section: Immunocytochemistry and Internalization Assaymentioning

confidence: 99%

Tumor cell-targeting by phage-displayed peptides

et al. 2002

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We isolated cancer cell -specific phages by subtracting and selecting complex peptide display phage libraries on cultured human cancer cells. The best candidate was selected by performing three rounds of subtraction before each of five selections on the human colorectal WiDr cell line. The phage showed more than 1000 -fold higher binding efficiency for WiDr cells when compared to five other human cancer cell lines, including two of colorectal origin, and when compared to wild -type M13 phage. Fifty -fold higher binding efficiency was also seen for a human breast cancer cell line. We show that the WiDr cell binding of the selected phage was efficiently competed by the synthetic peptide HEWSYLAPYPWF, predicted from the phage sequence. This confirms that the specificity of the peptide is independent of the display by the phage coat proteins. The identified peptide may target biomarkers linked to colorectal cancer, and thus be useful for designing gene transfer vectors as well as diagnostic and prognostic tools for this disease.

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“…The protocols of Poul et al 6 were followed with slight modifications. Cells were plated and grown to about 80% confluency on Lab -Tek chamber slides (eight wells; Nunc, Naperville, IL ).…”

Section: Immunocytochemistry and Internalization Assaymentioning

confidence: 99%

Tumor cell-targeting by phage-displayed peptides

et al. 2002

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“…Enrichment of ErbB2 phage over non-specific phage was 10-30 times higher when phage were recovered from within the cell compared to recovery from the cell surface, suggesting that cell selection specificity could be increased by recovering internalized phage. We confirmed this by applying this methodology to generate a panel of anti-tumor antibodies which were endocytosed into the breast tumor cell line SKBR3 as well as other tumor cells (Poul et al, 2000). Two of the specificities isolated included ErbB2 and transferrin receptor antibodies.…”

Section: Reportable Outcomesmentioning

confidence: 62%

Structural Basis of EGFR Dimerization for Drug Design

Marks¹

2000

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Public reporting burden lor this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project (0704-0188), Washington, DC 20503 AGENCY USE ONLY (Leave blank) REPORT DATE September 2000 REPORT TYPE AND DATES COVEREDFinal (01 Sep 97 -31 Aug 00) TITLE AND SUBTITLE Structural Basis of EGFR Dimerization for Drug Design AUTHOR(S)James Marks, M.D., Ph.D. FUNDING NUMBERS DAMD17-97-1-7250 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)The Resents of the University of California San Francisco, California 94143-0962 E-Mail: marksj@anesthesia.ucsf.edu 8. PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) U.S. Army Medical Research and Materiel CommandFort Detrick, Maryland 21702-5012 SUPPLEMENTARY NOTESReport contains color. ABSTRACT (Maximum 200 Words)Breast cancer occurs when there is a transition from normal breast epithelial cell behavior to that of uncontrolled cell growth. Cell surface receptors and specific growth factors play a crucial role in this transition. For this project, we proposed to evaluate the role of the epidermal growth factor receptor (EGFR) in breast cancer by expressing and purifying EGFR and solving the atomic structure. Antibodies generated from phage libraries would be used to: 1) facilitate structure solution, and 2) deliver drugs to EGFR expressing cells. EGFR was expressed at high levels and purified to homogeneity, but diffraction quality crystals were never obtained. As a result, efforts focused on antibody generation. A large panel (33) of human scFv antibodies to EGFR were isolated by selection on purified recombinant EGFR or on cells expressing EGFR. For cell selections, a method was developed which allowed direct selection from phage libraries of antibodies which trigger receptor mediated endocytosis. We show that this approach can be used either on tumor cell lines which overexpress the receptor or on cells transfected with the EGFR gene. This permits use of this methodology on transfected cells and provides a means of making antibodies without the need for protein expression and purification. We show that internalizing EGFR antibodies can be used to deliver cytotxic agents into the cytosol of EGFR expressing tumor cells by construction of immunoliposomes bearing an EGFR antibody on their surface. We are in the process of constructing and evaluating such an agent for breast cancer therapy. Where copyrighted material is quoted, permission has been obtained to use such material. SUBJECT ...

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“…For further studies we thus obtained the anti-erbB-2 scFv C6.5, which recognizes an ectodomain of the erbB-2 oncoprotein. 6 The cDNA of the anti-erbB-2 scFv was configured into a plasmid vector pSTCF allowing eukaryotic expression. This vector contains an Igk leader sequence, which routes heterologous proteins via the cellular secretory pathway.…”

Section: Resultsmentioning

confidence: 99%