IDENTIFICATION OF PARTIAL COMPLEMENTARY-DNA CLONES ENCODING A 59-KD PROTEIN WITH CHARACTERISTICS OF A UNIQUE ONCOFETAL ANTIGEN

Background: Oncofetal antigens (OFAs) are conserved tumor-associated a utoantigens or transplantation antigens present on the surface of all major classes of rodent and human tumors and on midgestational fetal c ells but not on normal neonatal or adult human and rodent tissues. A s yngeneically derived monoclonal antibody, MAb-115, recognizes murine O FAs of 44 and 200 kd in molecular mass. Purpose: Our goal was to clone and characterize the complementary DNAs (cDNAs) that encode these mur ine OFAs. Methods: Rabbit antiserum raised against purified 44-kd OFA glycoprotein was used to screen a mouse embryo cDNA-lambda phage expre ssion library. Recombinant phage clones positive for the expression of OFAs were detected by immunohistochemical staining, then isolated and plaque purified. The presence of an OFA-encoding sequence in the reco mbinant phage was confirmed by specific reaction of the expressed prot ein with MAb-115. Recombinant fusion protein was purified from the ext racts of corresponding lysogens. Rabbit antiserum against purified rec ombinant fusion protein was raised, and the capacity of this antiserum to detect the expression of OFA on rodent tumor and fetal cells was d etermined by flow cytometry. In addition, immunoreactivity of tumor be arer and hyperimmune murine sera to bacterially expressed recombinant OFA protein was evaluated by enzyme-linked immunosorbent assay. The OF A-expressing insert DNA from plaque-purified lambda clones was subclon ed into phagemid vectors for sequencing analysis. Results: Antiserum d erived against the isolated recombinant mouse embryo polypeptide mimic ked MAb-115 in its specific binding to all OFA-positive rodent tumor a nd fetal cell lines tested and likewise did not show reactivity to nor mal adult tissues. This antiserum specifically recognized the native 4 4- and 200-kd OFAs in extracts of murine lymphocytic lymphoma. Further more, sera of tumor-bearing mice or mice immunized with purified OFA o r intact, irradiated OFA-positive lymphocytic lymphoma cells also reac ted with the recombinant fusion protein. The characterization of the i solated clone included nucleotide sequence information followed by ana lysis of the deduced primary structure of the protein. Conclusions: Th ese data suggest that the isolated cDNA clones encode a distinct gene product which is widely expressed on the surface of tumor and fetal ce lls and represents the first characterized sequence of a true OFA. Imp lications: The availability of this cDNA, encoding a protein expressed only on tumor and fetal cells, provides a direct means to assess biol ogical characteristics of malignant tissue which can be assayed by bio chemical, histochemical, and molecular methods.