Identification of tumor‐associated antigens by screening phage‐displayed human cDNA libraries with sera from tumor patients

Abstract

Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage-display technology. We designed a new lambda vector to express protein fragments as N-terminal fusions to the D capsid protein and generated high-complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage-displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients. © 2003 Wiley-Liss, Inc. Key words: breast cancer; immune response; phage displayTumor-associated antigens recognized by humoral effectors of the immune system are an attractive target for diagnostic and therapeutic approaches to human cancer. A considerable number of this type of antigen have been identified over recent years through screening expression cDNA libraries from human solid tumors with sera of the autologous patients (SEREX, serologic identification of antigens by recombinant expression cloning). [1][2][3][4] This type of screening of a cDNA expression library by conventional methods requires the preparation of a large number of membrane filters blotted with bacteriophage plaques that are then searched with a specific probe. In the case of the SEREX experiments, the screening is performed using sera from cancer patients, which are usually available in very limited quantity. The second limitation is that such immunoscreening procedure does not allow selection of antigens that are recognized by sera from different patients.We tried to overcome these limitations and to simplify the screening procedure by performing affinity selection of cDNA libraries in very small volumes by combining the SEREX approach with that of phage-display technology. Phage-display technology is based on the insertion of foreign nucleotide sequences into genes encoding for various coat proteins of filamentous phage, resulting in a heterogeneous mixture of phages, each displaying the different peptide sequence encoded by a corresponding insert. A physical link between a displayed fusion protein and DNA encoded for it make this phage target selectable.Phage-display technology was introduced in 1985 by Smith 5 and has been widely used since for generating and screening peptide libraries to identify ligands for various kinds of receptor molecules. 6 -8 However, significantly less striking progress has been reported on the use of filamentous phage as display vector for constructing cDNA libraries. In most cases, foreign peptides are displayed on the filamentous phage capsid as N-terminal fusions to the major (pVIII) or the minor (pIII) coat protein. Large protein domains fused to pVIII disturb (with some rare...

Autoantibody Profiles Reveal Ubiquilin 1 as a Humoral Immune Response Target in Lung Adenocarcinoma

“…There is considerable evidence that the immune system produces an autoantibody response to neoplastic cells (16)(17)(18). The detection of such autoantibodies has been shown to have diagnostic and prognostic value (16,17,(19)(20)(21).…”

Chimeric Antigens ofToxoplasma gondii: Toward Standardization of Toxoplasmosis Serodiagnosis Using Recombinant Products

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