CLONING OF THE MURINE COUNTERPART OF THE TUMOR-ASSOCIATED ANTIGEN H-L6 - EPITOPE MAPPING OF THE HUMAN AND MURINE L6 ANTIGENS

The murine monoclonal antibody (mAb) L6 was raised against human lung carcinoma cells and found to recognize an antigen which is highly expr essed on lung, breast, colon, and ovarian carcinomas. Promising result s in phase 1 clinical studies with this antibody or its chimerized cou nterpart suggest the antigen recognized by mAb L6 (H-L6) is an attract ive target for monoclonal antibody-based cancer therapy. Further devel opment of L6 as an anti-tumor-targeting agent would benefit from the d evelopment of a murine model. However, initial attempts to develop suc h a model were hampered by our inability to generate antibodies agains t the murine homologue of the L6 antigen, M-L6. Here we describe the p reparation of the mAb 12A8, which was raised against murine thymic epi thelial cells, the tissue distribution of the murine antigen recognize d by 12A8, the cloning of a cDNA encoding the 12A8 target antigen, and the demonstration that this antigen is M-L6. Using H-L6/M-L6 chimeric proteins, we show that the region of the M-L6 protein recognized by m Ab 12A8 corresponds to the region of H-L6 recognized by mAb L6. There are five amino acid differences in the regions of the H-L6 and M-L6 pr oteins recognized by L6 and 12A8, respectively. We further mapped the protein epitope recognized by L6 by individually exchanging each of th ese residues in H-L6 with the corresponding residue found in M-L6. Sub stitution of the single H-L6 residue Leu122 with Ser resulted in the H -L6 mutant HL6-L122S which failed to bind L6. The HL6-L122S mutant als o failed to bind 12A8. Substituting residue Ser122 in M-L6 with Leu di d not prevent 12A8 binding and did not result in L6 binding. The avail ability of mAb 12A8 and the finding that it recognizes the same region of M-L6 that is recognized by L6 on H-L6 might allow the development of a murine tumor model in which the L6 antigen can be further evaluat ed as a therapeutic target.