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 expressed on lung, breast, colon, and ovarian carcinomas. Promising results in phase 1 clinical studies with this antibody or its chimerized counterpart suggest the antigen recognized by mAb L6 (H-L6) is an attractive target for monoclonal antibody-based cancer therapy. Further development of L6 as an anti-tumor-targeting agent would benefit from the development of a murine model. However, initial attempts to develop such a model were hampered by our inability to generate antibodies against the murine homologue of the L6 antigen, M-L6. Here we describe the preparation of the mAb 12A8, which was raised against murine thymic epithelial cells, the tissue distribution of the murine antigen recognized 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 mAb 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 proteins recognized by L6 and 12A8, respectively. We further mapped the protein epitope recognized by L6 by individually exchanging each of these residues in H-L6 with the corresponding residue found in M-L6. Substitution 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 also failed to bind 12A8.(ABSTRACT TRUNCATED AT 250 WORDS)
Edwards
C P
CP
Department of Biological Structure, University of Washington School of Medicine, Seattle 98195, USA.
Farr
A G
AG
Marken
J S
JS
Nelson
A
A
Bajorath
J
J
Hellström
K E
KE
Hellström
I
I
Aruffo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018076:DNA, Complementary; D000939:Epitopes; D009363:Neoplasm Proteins; C074075:TM4SF1 protein, human