CYTOTOXIC AND ANTITUMOR-ACTIVITY OF A RECOMBINANT TUMOR NECROSIS FACTOR-B1(FV) FUSION PROTEIN ON LE(Y) ANTIGEN-EXPRESSING HUMAN CANCER-CELLS

We have constructed a fusion protein composed of tumor necrosis factor alpha (TNF-alpha) fused at its COOH terminus to the scFv region of mo noclonal antibody (mAb) B1, an antibody that recognizes Le(Y) antigen present on many human cancer cells, Our rationale for fusing the scFv to the COOH terminus of TNF was to diminish the binding of the fusion protein to TNF receptors because the COOH terminus of TNF is involved in binding, and thus to partially inactivate (detoxify) the molecule. The Fv region should then target and accumulate the fusion protein on cancer cells, which should compensate for the reduced binding affinity of the TNF moiety and lead to selective killing of TNF-sensitive anti gen-expressing cancer cells. The fusion protein was expressed in Esche richia coli and found in insoluble inclusion bodies, After refolding a nd purification by anion exchange, Ni-NTA affinity, and size-exclusion chromatography, we obtained monomeric TNF-B1(Fv). This molecule binds to Le(Y) antigen on cancer cells with the same affinity as B1(scFv) a nd B1(scFv) immunotoxins but with significantly lower affinity to the TNF receptor compared to the TNF trimer. TNF-B1(Fv) is very toxic to L e(Y) antigen-expressing cancer cells that are sensitive to TNF (e.g., MCF-7 breast or CRL-1739 gastric cancer cells). This cytotoxicity is a ntibody targeted and TNF mediated because it can be prevented (as show n on MCF-7 cells) by an antibody competing for Le(Y) antigen binding a nd by an antibody that neutralizes TNF-alpha. TNF-B1(FV) kills TNF-alp ha-sensitive cells that do not express the target antigen only at much higher doses than TNF trimer, and it does not kill Le(Y)-bearing but TNF-alpha-resistant cells. TNF-B1(Fv) can cause significant tumor regr ession of MCF-7 tumor xenografts in mice at doses that are not toxic t o the mice. Thus, the reduced binding of the TNF moiety to TNF recepto rs, combined with binding of the B1(Fv) portion to Le(Y) antigen, make s TNF-B1(Fv) an agent for selective killing of Le(Y)-expressing TNF-se nsitive cancer cells.