Mapping and regulation of the tumor-associated epitope recognized by monoclonal antibody RS-11

We have previously described a rat monoclonal antibody, RS-ll, which recogn izes a tumor-associated antigen common to several species. In the present s tudy, we have cloned and characterized the antigen recognized by RS-11, We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-ll, DNA sequence analysis revealed that this cl one contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides b ased on this DNA sequence analysis and examined their reactivity with RS-ll to accurately map the RS-ll epitope. We determined that the epitope reside s within a region of seven amino acids on the alpha-helix 2B domain of kera tin 18 in which two amino acids (Leu(366) and Lys(370)) are completely cons erved among intermediate filaments as well as other keratin members that ar e immunoreactive with RS-ll, These two residues are sequentially discontinu ous but spatially adjacent, The RS-ll epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-1 1 is upregulated by malignant transformation or stimulation with either epi dermal growth factor or transforming growth factor alpha.