| Abstrakt: |
Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have engineered and selected a completely human antibody to epithelial glycoprotein-2 (EGP-2), a transmembrane glycoprotein present on virtually all human simple epithelia and abundantly expressed on a variety of human carcinomas. We chose to use the procedure of “guided selection” to rebuild a high-affinity murine antibody into a human antibody, using two consecutive rounds of variable domain shuffling and phage library selection. As a starting antibody, the murine antibody MOC-31 was used. After the first round of guided selection, where the VH of MOC-31 was combined in Fab format with a human VLCL library, a small panel of human light chains was identified, originating from a segment of the VκIII family, whereas the MOC-31 VL is more homologous to the VκII family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the VL of C3 with a human VH library, while retaining the VH CDR3 of MOC-31, clones were selected using human VH genes originating from the rarely used VH7 family. The best clone, 9E, shows over 13 amino acid mutations from the germline sequence, has an off-rate comparable to the original antibody and specifically binds to the “MOC-31“-epitope on EGP-2 in specificity and competition ELISA, FACS analysis and immunohistochemistry. In both VL and VH of antibody 9E, three germline mutations were found creating the MOC-31 homologue residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in VH CDR2, is likely to be involved in antigen binding. We conclude that, although they may bind the same epitope and have similar binding affinity to the antigen as the original murine antibody, human antibodies derived by guided selection unlike CDR-grafted antibodies, may retain only some of the original key elements of the binding site chemistry. The selected human anti-EGP-2 antibody will be a suitable reagent for tumour targeting. |
Full Text from ScienceDirect