ABSTRACT
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.
Subject(s)
Breast Neoplasms/metabolism , Cell Adhesion Molecules/metabolism , Endocytosis , Lutheran Blood-Group System/metabolism , Animals , Biomarkers, Tumor , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Cytotoxicity Tests, Immunologic , Female , Humans , Immunoconjugates/pharmacology , Immunoglobulin Fc Fragments/immunology , Immunohistochemistry , Lutheran Blood-Group System/genetics , Mice , Protein Binding , Protein Transport , Single-Chain Antibodies/pharmacologyABSTRACT
Laminin-511, a major component of endothelial basement membrane, consists of α5, ß1, and γ1 chains. The short arm region of the α5 chain is a structural feature of endothelial laminins. In this study, we identified active sequences for human umbilical vein endothelial cells (HUVECs) using recombinant proteins and synthetic peptides. The short arm of the α5 chain contains three globular domains [laminin N-terminal globular domain, laminin 4 domain a, and laminin 4 domain b (LN, L4a, and L4b)] and three rod-like elements [laminin epidermal growth factor-like domain a, b, and c (LEa, LEb, and LEc)]. The cell attachment assay using recombinant proteins showed that RGD-independent cell attachment sites were localized in the α5LN-LEa domain. Further, we synthesized 70 peptides covering the amino acid sequences of the α5LN-LEa domain. Of the 70 peptides, A5-16 (mouse laminin α5 230-243: LENGEIVVSLVNGR) potently exhibited endothelial cell attachment activity. An active sequence analysis using N-terminally and C-terminally truncated A5-16 peptides showed that the nine-amino acid sequence IVVSLVNGR was critical for the endothelial cell attachment activity. Cell adhesion to the peptides was dependent on both cations and heparan sulfate. Further, the A5-16 peptide inhibited the capillary-like tube formation of HUVECs with the cells forming small clumps with short tubes. The eight-amino acid sequence EIVVSLVN in the A5-16 peptide was critical to inhibit HUVEC tube formation. This amino acid sequence could be useful for grafts and thus modulate endothelial cell behavior for vascular surgery. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Subject(s)
Laminin/chemistry , Peptides/chemistry , Amino Acid Sequence , Animals , Cell Adhesion/physiology , Endothelial Cells , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Mice , Recombinant Proteins/chemistryABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Cell Surface Display Techniques , Glycoproteins/immunology , Lutheran Blood-Group System/immunology , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Cell Line, Tumor , Cell Movement/drug effects , Enzyme-Linked Immunosorbent Assay , Epitope Mapping/methods , Epitopes/chemistry , Epitopes/immunology , Humans , Peptide Library , Protein Binding/immunology , Protein Interaction Domains and Motifs/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/pharmacologyABSTRACT
Epithelial cells, both normal and precancerous, stably anchor to basement membranes, whereas malignant tumors pass through them to achieve metastasis. Of basement membrane components, laminin-511 (α5, ß1, γ1; LM-511) has been found to be a major isoform in many adult basement membranes. Several studies have shown that LM-511 promotes not only cell adhesion but also tumor cell migration. Thus, LM-511 can be viewed like two distinct molecules in normal vs. tumor cells; tumor cells seem to be able to alter their response (adhesive vs. migratory) to LM-511. In this study we examined the effects of biologically active molecules on A549 lung adenocarcinoma cell adhesion to LM-511. Of them, phorbol 12-myristate 13-acetate (PMA) induced transition to a rounded cell shape and significantly promoted cell migration on LM-511. The attachment of PMA-treated A549 cells to LM-511 was weaker than that of control cells. PMA-stimulated signaling pathway reduced the binding of integrin α3ß1 to LM-511. Cell migration assays using inhibitors for signal transduction and cytoskeletal organization showed that suppression of cell adhesion via the rho-associated protein kinase (ROCK) pathway promoted tumor cell migration on LM-511. Our results suggest that the ROCK pathway is involved in the transition from static to migratory cell behaviors on LM-511.
