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With the aim of identifying tumor-associated antigens that could be potential markers and/or targets of diagnostic and/or therapeutic approaches, we studied the occurrence of circulating IgG antibodies to human stromelysin-3, collagenase-3, galectin-3 and mesothelin, by Western blot against their purified recombinant forms, in the sera of 50 patients with pharynx/larynx squamous cell carcinoma (PLSCC), as well as in the sera of 50 healthy blood donors. Overall, antibodies to collagenase-3 were detected in 50% of all the cancer patients and 16% of the blood donors examined; this percentage difference was statistically significant (p = 0.00066). With respect to anti-galectin-3 antibodies, the percentages were 32% and 18%, respectively, but they were not statistically different (p = 0.16). Low levels of antibodies to stromelysin-3 and to mesothelin were detected in sera from only two cancer patients. No significant correlations were found in the present study between the presence of antibodies to these proteins and tumor site, clinical and T stages, lymph node involvement, DNA ploidy and histological grade of differentiation of the primary tumors. To our knowledge, this is the first report on the detection of circulating IgG to collagenase-3 in cancer patients. Some of the percentages found here in certain groups of patients are among the highest reported of circulating antibodies to any tumor component studied so far. The monitoring and the use of human antibodies to collagenase-3 could be of diagnostic and therapeutic interest.

Background: Generation and cloning of antibodies against cell surface antigens can be simplified by combining DNA immunization which enables generation of antibodies against a protein in its natural configuration without the need for any protein purification step and antibody phage display which due to its immense screening power and physical coupling between the phenotype and genotype of antibodies simplifies the cloning of antibody genes., Objectives: Since DNA immunization is expected to elicit antibodies against a protein in its natural configuration, we wanted to see if it can mimic the antibody response generated by cell immunization., Study Design: A phage display library made from splenic mRNA of a mouse immunized with mesothelin cDNA was panned on mesothelin-positive cells. The single-chain Fvs (scFvs) selected were then analyzed., Results: We obtained several anti-mesothelin scFvs. One of these Fvs is almost identical to the Fv of a monoclonal antibody that was previously obtained from a hybridoma in which the mice were immunized with a mesothelin-positive ovarian cancer cell line. Another Fv was found to be specific for mesothelin present on human cells., Conclusion: Our results indicate that an antibody phage display library made from spleens of DNA-immunized mice is a rapid and efficient alternative to cell immunization for obtaining antibodies against different epitopes of a membrane antigen that is very difficult to purify in a native form.

Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers. Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents. In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin. When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice. After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells. The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A. The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37 degrees C and is very cytotoxic to cells expressing mesothelin. It also produces regressions of tumors expressing mesothelin. This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent. This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.

Phage display has been used to select single-chain Fvs (scFvs) against mesothelin, a surface antigen present on mesothelial cells as well as mesotheliomas and non-mucinous ovarian cancers. Several attempts to produce anti-mesothelin hybridomas from spleen cells of mice immunized with recombinant mesothelin were unsuccessful. This report describes the isolation of anti-mesothelin scFvs from a phage display library made from the mRNA of the same spleens. Panning on recombinant antigen produced in E. coli or on antigen positive cells was employed. Several scFvs which bind specifically to mesothelin were isolated. Panning on recombinant antigen yielded five different scFvs. Panning on cells selected two different scFvs which also differ from the scFvs selected by recombinant antigen. The heavy chains of the scFvs selected on recombinant antigen are derived from four different heavy chain gene families and the scFvs selected on cells are derived from two of these families. In contrast, the light chains of all of these scFvs are derived from family XI. Moreover, the light chains of the two scFvs selected on cells are very similar to the light chains of two of the scFvs selected by panning on recombinant mesothelin except for a few point mutations. One of these scFvs which have been studied in detail has been shown to bind specifically to mesothelin positive transfected cells.