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

Abstract

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 × 10⁷κ 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.

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