title: Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody.
abstract: 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.
authors.affiliation: Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, 16132 Genova, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Piazza
authors.forename: Emanuela
authors.initials: E
authors.lastname: Cha
authors.forename: Italia
authors.initials: I
authors.lastname: Bongarzone
authors.forename: Silvana
authors.initials: S
authors.lastname: Canevari
authors.forename: Andrea
authors.initials: A
authors.lastname: Bolognesi
authors.forename: Letizia
authors.initials: L
authors.lastname: Polito
authors.forename: Antonio
authors.initials: A
authors.lastname: Bargellesi
authors.forename: Francesca
authors.initials: F
authors.lastname: Sassi
authors.forename: Silvano
authors.initials: S
authors.lastname: Ferrini
authors.forename: Marina
authors.initials: M
authors.lastname: Fabbi
mesh_terms: D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000032:analysis / Q000276:immunology* / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D002450:Cell Communication / Q000276:immunology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002966:Clathrin / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D008024:Ligands; D010940:Plant Proteins / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000276:immunology; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D020410:Activated-Leukocyte Cell Adhesion Molecule; D000906:Antibodies; D002966:Clathrin; D007128:Immunoglobulin Fragments; D008024:Ligands; D010940:Plant Proteins; D011994:Recombinant Proteins