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Isolation of Human Antibodies against the Central DNA Binding Domain of p53 from an Individual with Colorectal Cancer Using Antibody Phage Display

Department of Medical Oncology, St. Vincent’s Hospital, Sydney, 2010, NSW, Australia

Purpose: Serum anti-p53 antibodies are detected in 30% of patients with cancer, and most of these antibodies are directed against the NH₂- and COOH-terminal domains of p53. The aim of this study was to identify individuals with serum reactivity against the central and COOH-terminal domains of p53 and to isolate and characterize these recombinant human antibodies.

Experimental Design: The serum of individuals with colorectal cancer was screened for the presence of anti-p53 antibodies. Antibody phage display libraries were constructed from four immunoreactive individuals, and the libraries were panned against the central and COOH-terminal domains of p53.

Results: An antibody fragment (1159.8) that was specific for the whole molecule as well as the central domain was isolated from one of the libraries. The serum from the original individual inhibited the binding of this Fab fragment to p53, thus indicating that the antibody reflected the specificity of the in vivo immune response. The VL and VH genes from Fab 1159.8 matched germ-line genes from the VH3 and VK2 families, respectively. Competition analysis with monoclonal antibodies showed that Fab binding could be inhibited most effectively with DO11 and, to a lesser extent, Pab240, indicating an epitope within or adjacent to residues 181–190 of p53.

Conclusions: The successful isolation of this human anticentral domain Fab provides additional insight into the nature and specificity of the tumor-specific immune response. This antibody could be used as a reagent for functional studies of p53, or it may be a candidate for use as an anticancer idiotypic vaccine.

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