An Anti-Human Lutheran Glycoprotein Phage Antibody Inhibits Cell Migration on Laminin-511: Epitope Mapping of the Antibody

Abstract

The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is an Ig superfamily (IgSF) transmembrane receptor for laminin α5. Although Lu is not present in normal hepatocytes, its expression is significantly increased in hepatocellular carcinoma (HCC). In this study, we isolated thirteen phage antibodies to Lu from a phage library of peripheral blood from HCC patients, suggesting that these patients produced autoantibodies against endogenous Lu. To characterize the phage antibodies, we determined the Lu domains they recognize. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of one phage antibody (A7) was localized to the D5 domain. The other phage antibodies recognized the D2 domain, which is also recognized by a function blocking mouse monoclonal antibody. One of the antibodies to D2 (C7) inhibited the binding of Lu to ligand, and it also prevented tumor cell migration on laminin-511 (LM-511). However, the C7 scFv purified from the periplasm fraction of bacteria did not exhibit the inhibitory effects, indicating that the scFv form could not sterically inhibit the binding of Lu to LM-511. We also identified the amino acid residues that form the epitope recognized by the C7 phage antibody. Mutagenesis studies showed that Arg247 is necessary for forming the epitope. The C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression and/or metastasis.

Dimerization and phosphorylation of Lutheran/basal cell adhesion molecule are critical for its function in cell migration on laminin

“…Our results on Lu-mediated migration are in accordance with previous studies performed with human fibrosarcoma cell lines overexpressing Lu or with hepatocellular carcinoma cells (29,40). Lu expression has also been shown to mediate motility on biliary cells in a liver injury context (27).…”

Angiotropism, pericytic mimicry and extravascular migratory metastasis: an embryogenesis-derived program of tumor spread

“…(iii) Laminin-332 plays a role in the pulmonary metastases of breast carcinoma and may provide a target for anti-metastatic therapy [50]. (iv) Lu/BCAM functions as the receptor for specific ligand laminin-α5 chain and is involved in cell-cell adhesion and migration in several malignancies [103,104]. This laminin receptor may have potential as a novel therapeutic target [103,104].…”

Immunotoxin Screening System: A Rapid and Direct Approach to Obtain Functional Antibodies with Internalization Capacities

“…The notion was also illustrated for generation of ADC by using a human single chain variable fragment (scFv)-Fc antibody against CD239/basal cell adhesion molecule for treatment of breast cancer [ 76 ]. The scFV-Fc antibody termed C7-Fc, originally identified from screening the scFv phage libraries [ 77 ], was bound to DT3C to characterized its internalization property, and selectively target and kill SKBR3 breast cancer cells. This work also provides evidence supporting the application of our cell-based IT screening system to characterize the candidates identified from the phage display libraries.…”