A novel tumor-specific human single-chain Fv selected from an active specific immunotherapy phage display library

A colon tumor-associated antigen, CTAA 28A32-32K (CTA # 2E), related to the annexin family of proteins, was initially identified by its reactivity with a low affinity human IgM monoclonal antibody (mAb), 28A32. Both in vitro lymphoproliferative assays with human peripheral blood lymphocytes and delayed type hypersensitivity responses in patients immunized with autologous colon tumor cells indicated that CTA # 2E elicits potent T cell mediated responses and may be an important antigen in the development of a generic colorectal vaccine (Pomato et al. Vaccine Res 1994;3:145-161). A CTA # 2E-specific, murine hybridoma-derived mAb, 5-11A, which recognizes the amino-terminus of the tumor-associated antigen, exhibited qualitative human colon tumor-specific immunohistochemical reactivity. To rapidly develop a human mAb with similar antigen specificity and tumor reactivity as the murine 5-11A mAb, antibody phage display technology was employed. Two human antibody phage display libraries with 3.1 x 10(7) and 2.3 x 10(8) members were prepared from the variable region genes expressed by circulating B cells of patients undergoing active specific immunotherapy (ASI) with autologous tumor cells, predominantly from the colon, admixed with Bacille Calmette-Guerin (BCG). A CTA # 2E-reactive human single-chain (sc)Fv was selected by panning the larger library on decreasing concentrations of biotinylated tumor-associated antigen in solution. It exhibited similar antigen specificity as the murine hybridoma-derived 5-11A scFv, requiring the presence of the CTA # 2E amino-terminus for reactivity. This human scFv exhibited qualitative human colon tumor-specific immunohistochemical reactivity when displayed as a gene III fusion protein on phage. When reconstructed and expressed as an intact human IgG1, K mAb, its qualitative colon tumor-specificity was unaltered. Two other CTA # 2E-reactive human scFvs were selected from the smaller library by panning initially on decreasing concentrations of CTA # 2E coated to polystyrene and then on biotinylated CTA # 2E in solution. These human scFvs, which exhibited modest reactivity with different epitopes on the CTA # 2E antigen, did not exhibit human colon tumor-specific immunohistochemical reactivity.