Antibody–drug conjugates (ADCs) are attractive in cancer therapy because they can directly bind to cancer cells and provide anticancer activity. To kill cancer cells with ADCs, the target antigens are required not only to be highly and/or selectively expressed on cancer cells but also internalized by the cells. CD239, also known as the Lutheran blood group glycoprotein (Lu) or basal cell adhesion molecule (B-CAM), is a specific receptor for laminin α5, a major component of basement membranes. Here, we show that CD239 is strongly expressed in a subset of breast cancer cells and internalized into the cells. We also produced a human single-chain variable fragment (scFv) specific to CD239 fused with human IgG1 Fc, called C7-Fc. The binding affinity of the C7-Fc antibody is similar to that of mouse monoclonal antibodies. Although the C7-Fc antibody alone does not influence cellular functions, when conjugated with a fragment of diphtheria toxin lacking the receptor-binding domain (fDT), it can selectively kill breast cancer cells. Interestingly, fDT-bound C7-Fc shows anticancer activity in CD239-highly positive SKBR3 cells, but not in weakly positive cells. Our results show that CD239 is a promising antigen for ADC-based breast cancer therapy.

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.