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

Abstract

SUMMARYAn immunoglobulin phage display library constructed from a tumour-associated pericolic lymph node was panned against the extracellular domain of the oncoprotein c-erbB-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-erbB-2 over-expressing breast cancer cell line SKBR3 but not to the lowexpressing cell line MDA-MB-231. We conclude that a single lymph node library of moderate diversity (2 × 10 7 k light chain and g heavy chain clones), when derived from an individual whose colorectal tumour over-expressed c-erbB-2, can be successfully panned to isolate a number of unique Fabs specific for this antigen. The nature of the anti-c-erbB-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.

Immune responses to all ErbB family receptors detectable in serum of cancer patients

“…Several studies have previously established that patients with tumors overexpressing the ErbB2 protein, can spontaneously develop natural immunity to this receptor in the apparent absence of structural abnormalities (Pupa et al, 1993;Disis et al, 1994aDisis et al, ,b, 1997Fisk et al, 1995;Peoples et al, 1995;Clark et al, 1997). In the present study, we extend this observation by demonstrating the presence of speci®c antibody responses against all ErbB receptors including EGFR, ErbB2, ErbB3 and ErbB4 in tumor patients with di erent epithelial malignancies.…”

“…Humoral responses directed against the EGFR have been reported (Glushkov et al, 1996). Moreover, it has been reproducibly documented that some breast and ovarian tumor patients mount natural T and B cell responses to ErbB2 (Pupa et al, 1993;Disis et al, 1994aDisis et al, ,b, 1997Fisk et al, 1995;Peoples et al, 1995;Clark et al, 1997). This receptor has triggered particular interest based upon evidence that its overexpression independently predicts a worse disease course in lymph node positive breast cancer (Slamon et al, 1989).…”

Antigen-Driven Oligoclonal Expansion of Tumor-Infiltrating B Cells in Infiltrating Ductal Carcinoma of the Breast

“…Breast cancer-reactive B cells have also been identified in regional or draining lymph nodes of breast cancer patients (for examples, see Refs. [12][13][14][15]. However, little direct evidence exists that TIL-B in breast cancer are tumor specific, although tumor cell-reactive TIL-B have been cloned from other tumor types, including melanoma, colon carcinoma, ovarian carcinoma, lung carcinoma, glioma, sarcoma, neuroblastoma, and Hodgkin's lymphoma (16 -23).…”

Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display

“…13,14 Alternatively, it may also be exploited to study in vivo humoral immune responses to particular antigens. 5,15,16 We set out to study the humoral immune response in a colorectal cancer (CRC) patient by screening phage antibody repertoires made from patient material for the presence of antibodies reactive with colon tumors. Since lymph node tissue has proven to be a valuable source of variable (V)-genes for the construction of phage antibody repertoires 17 and since this tissue has also been shown to be a rich source of antibodies to known tumor antigens, 5 we decided to use patient-derived lymph nodes (LN) draining a CRC for the construction of "immune" phage antibody repertoires.…”