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First published online 15 March 2005
doi: 10.1242/jcs.02280

Journal of Cell Science 118, 1515-1525 (2005)
Published by The Company of Biologists 2005
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Research Article

Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody

Tiziana Piazza1, Emanuela Cha2, Italia Bongarzone3, Silvana Canevari3, Andrea Bolognesi4, Letizia Polito4, Antonio Bargellesi5, Francesca Sassi1, Silvano Ferrini1,* and Marina Fabbi1

1 Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, 16132 Genova, Italy
2 Centro Biotecnologie Avanzate, Largo R. Benzi 10, 16132 Genova, Italy
3 Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italy
4 Department of Experimental Pathology, University of Bologna, Via S. Giacomo 14, 40126 Bologna, Italy
5 Department of Experimental Medicine, University of Genova, Via L. B. Alberti 2, 16132 Genova, Italy

* Author for correspondence (e-mail: silvano.ferrini{at}istge.it)

Accepted 24 January 2005

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, promotes heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. Here we describe a fully human single-chain antibody fragment (scFv) directed to ALCAM/CD166. We selected the I/F8 scFv from a phage display library of human V-gene segments by cell panning and phage internalization into IGROV-I human ovary carcinoma cells. The I/F8 specificity was identified as ALCAM/CD166 by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) peptide mass fingerprinting of the I/F8-immunoprecipitated protein. The I/F8 scFv reacts with the human, monkey and murine ALCAM/CD166 molecule, indicating that the recognized epitope is highly conserved. The I/F8 scFv completely abolished binding of both ALCAM/Fc and CD6/Fc soluble ligands, whereas it did not compete with the anti-ALCAM/CD166 murine monoclonal antibodies J4-81 and 3A6 and therefore recognizes a different epitope. Engagement through I/F8 scFv, 3A6 monoclonal antibody or CD6/Fc ligand induced ALCAM/CD166 internalization, with a kinetics slower than that of transferrin in the same cells. Newly internalized I/F8-ALCAM complexes colocalized with clathrin but not with caveolin and we demonstrated, using surface biotinylation and recycling assays, that endocytosed ALCAM/CD166 recycles back to the cell surface. Such an endocytic pathway allows the efficient delivery of an I/F8 scFv-saporin immunotoxin into tumor cells, as the conjugates are able to selectively kill cell lines expressing ALCAM/CD166. Altogether these data provide evidence of the suitability of the I/F8 scFv for further functional analysis of ALCAM/CD166 and intracellular delivery of effector moieties.

Key words: Recombinant antibodies, ALCAM/CD166, Endocytosis, Recycling




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