Cytotoxic and antitumor activity of a recombinant tumor necrosis factor-B1(Fv) fusion protein on LeY 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 monoclonal antibody (mAb) B1, an antibody that recognizes LeY antigen present on many human cancer cells. Our rationale for fusing the scFv to the COOH terminus o...
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Veröffentlicht in: | Clinical cancer research 1996-09, Vol.2 (9), p.1523-1531 |
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Zusammenfassung: | We have constructed a fusion protein composed of tumor necrosis factor alpha (TNF-alpha) fused at its COOH terminus to the
scFv region of monoclonal antibody (mAb) B1, an antibody that recognizes LeY 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 antigen-expressing cancer cells. The fusion protein
was expressed in Escherichia coli and found in insoluble inclusion bodies. After refolding and purification by anion exchange,
Ni-NTA affinity, and size-exclusion chromatography, we obtained monomeric TNF-B1(Fv). This molecule binds to LeY antigen on
cancer cells with the same affinity as B1(scFv) and B1(scFv) immunotoxins but with significantly lower affinity to the TNF
receptor compared to the TNF trimer. TNF-B1(Fv) is very toxic to LeY antigen-expressing cancer cells that are sensitive to
TNF (e.g., MCF-7 breast or CRL-1739 gastric cancer cells). This cytotoxicity is antibody targeted and TNF mediated because
it can be prevented (as shown on MCF-7 cells) by an antibody competing for LeY antigen binding and by an antibody that neutralizes
TNF-alpha. TNF-B1(Fv) kills TNF-alpha-sensitive cells that do not express the target antigen only at much higher doses than
TNF trimer, and it does not kill LeY-bearing but TNF-alpha-resistant cells. TNF-B1(Fv) can cause significant tumor regression
of MCF-7 tumor xenografts in mice at doses that are not toxic to the mice. Thus, the reduced binding of the TNF moiety to
TNF receptors, combined with binding of the B1(Fv) portion to LeY antigen, makes TNF-B1(Fv) an agent for selective killing
of LeY-expressing TNF-sensitive cancer cells. |
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ISSN: | 1078-0432 1557-3265 |