Isolation of human anti-c-erbB-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-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 low-expressing cell line MDA-MB-231. We conclude that a single lymph node library of moderate diversity (2 x 10(7) kappa light chain and gamma 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.
Clark
M A
MA
Department of Medical Oncology, St Vincent's Hospital, Darlinghurst, Australia.
Hawkins
N J
NJ
Papaioannou
A
A
Fiddes
R J
RJ
Ward
R L
RL
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003106:Colon / Q000276:immunology; D015179:Colorectal Neoplasms / Q000235:genetics / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D005098:Exotoxins / Q000276:immunology; D015723:Gene Library; D018734:Genes, erbB-2; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007136:Immunoglobulins / Q000276:immunology; D007150:Immunohistochemistry; D007328:Insulin / Q000276:immunology; D008198:Lymph Nodes / Q000276:immunology; D008969:Molecular Sequence Data; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA; D012710:Serum Albumin, Bovine / Q000276:immunology; D013745:Tetanus Toxoid / Q000276:immunology; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D005098:Exotoxins; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D007328:Insulin; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D012710:Serum Albumin, Bovine; D013745:Tetanus Toxoid; C101006:HAR-TX beta 2 protein, recombinant