Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent

Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have en gineered and selected a completely human antibody to epithelial glycoprotei n-2 (EGP-2), a transmembrane glycoprotein present on virtually all human si mple epithelia and abundantly expressed on a variety of human carcinomas. W e chose to use the procedure of "guided selection" to rebuild a high-affini ty murine antibody into a human antibody, using two consecutive rounds of v ariable domain shuffling and phage library selection. As a starting antibod y, the murine antibody MOC-31 was used. After the first round of guided sel ection, where the V-H of MOC-31 was combined in Fab format with a human VLC L library, a small panel of human Light chains was identified, originating from a segment of the V kappa III family, whereas the MOC-31 V-L is more ho mologous to the V kappa II family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the V-L of C3 with a human V-H library, while retaining the V-H CDR3 of MOC-31, clones we re selected using human V-H genes originating from the rarely used V(H)7 fa mily. The best clone, 9E, shows over 13 amino acid mutations from the germl ine sequence, has an off-rate comparable to the original antibody and speci fically binds to the "MOC-31"-epitope on EGP-2 in specificity and competiti on ELISA, FAGS analysis and immunohistochemistry. In both V-L and V-H of an tibody 9E, three germline mutations were found creating the MOC-31 homologu e residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in V-H CDR2, is likely to be invol ved in antigen binding. We conclude that, although they may bind the same e pitope and have similar binding affinity to the antigen as the original mur ine antibody, human antibodies derived by guided selection unlike CDR-graft ed antibodies, may retain only some of the original key elements of the bin ding site chemistry. The selected human anti-EGP-2 antibody will be a suita ble reagent for tumour targeting. (C) 2000 Academic Press.