Isolation of human anti‐c‐ erb B‐2 Fabs from a lymph node‐derived phage display library

An immunoglobulin phage display library constructed from a tumour‐associated pericolic lymph node was panned against the extracellular domain of the oncoprotein c‐ erb B‐2. Sixteen independent clones were confirmed as positive binders based on ELISA analysis of soluble Fabs. Nucleotide sequencing demonstrated that the V H region of 12 clones belonged to four different V gene families, and the clones demonstrated varying degrees of somatic mutation compared with germ‐line sequences. Fab fragments were examined for cross‐reactivity by ELISA and shown to be negative against a panel of irrelevant self and non‐self antigens, including bovine serum albumin (BSA), mouse immunoglobulin, tetanus toxoid, heregulin‐PE40‐FLAG and insulin. Reactivity of Fabs in vitro was verified by immunocytochemistry, which showed binding to the c‐ erb B‐2 over‐expressing breast cancer cell line SKBR3 but not to the low‐expressing cell line MDA‐MB‐231. We conclude that a single lymph node library of moderate diversity (2 × 10 7 κ light chain and γ heavy chain clones), when derived from an individual whose colorectal tumour over‐expressed c‐ erb B‐2, can be successfully panned to isolate a number of unique Fabs specific for this antigen. The nature of the anti‐c‐ erb B‐2 Fabs recovered from this library suggests that they may have resulted from a humoral immune response in the individual, and that in vivo antibody responses to tumour‐associated antigens may be exploited in vitro for the production of tumour‐specific recombinant antibodies.