Specific Antitumor Activity Of Recombinant Antibody/Granzyme B

Gene therapy has been widely used to cure cancers in the past decades and provides a promising way to cancer therapy. It could be reasonable to suppress tumor growth by inducing tumor cells to die. This active antitumor strategy, however, demands specificity, efficiency, persistence and minimal side-effect.Granzyme B(GrB) is member of serine proteinase family and plays an essential role in CTL/NK-mediated killing. Redundancy of GrB-mediated death pathways enables lymphocytes to eliminate unwanted cells efficiently. Therefore, GrB could be a new good candidate to kill tumor cells.Our results showed ectopic expression of active GrB(GrBa) led cells to death. This was probably because GrBa protein was overexpressed in cytoplasm and its serine proteinase activity caused proteolysis of endogenous substrates which might be important to cellular structure and function. There was a special phenomenon that cells, when subjected to GrBa-mediated death, appeared as volume extension and often in the wake of multiple nuclei. These giant cells with multilobed or multiple nuclei exhibited mitotic and cytoskeletal abnormality.In present study, the gene of single chain antibody against HER-2, a kind of tumor marker on membrane, was fused to 5'-end of GrBa gene. Sequence encoding Pseudomonas exotoxin A(PE) translocating peptide was localized between antibody gene and GrBa gene. The resulting fusion protein gene was designated as irnmunoGrB gene. In theory, on entry into endosome of tumor cells irnmunoGrB protein releases C-terminal PE II-GrBa into cytosol as a result of auto-cleavage of peptide bond between Arg279 and Gly280. Two kinds of PE translocating peptides covering 253-364 aa and 253-358 aa were compared for their endosome-disruptive function. On one hand, it was demonstrated that presence of part of PE II sequence(280-364 aa and 280-358 aa) hardly abrogated GrBa activity to induce cell death, which was proven by the data from transient expression, inducible expression and constitutive expression as well, and that the shorter N-terminal peptide PE II-GrBa fusion protein had, the closer extent of enzyme activity and cell growth inhibition to that of GrBa alone. On the other hand, immunoGrB was found inactive in transient and stable transfection research until when translocating from endosome in HER-2 positive tumor cells, it turned into an active form. We next tested antitumor activity of immunoGrB in nude mouse models. It was observed that intramuscle administration of immunoGrB gene suppressed HER-2 positive tumor.Genetically modified lymphocytes strategy was further tested hi cultured Jurkat cells. ImmunoGrB transduced Jurkat cells produced and secreted GrBa fusion protein while exhibiting a normal growth curve. By co-cultivation, these modified Jurkat cells were shown to have selective cytotoxicity to HER-2 positive tumor cells. Over 70% killing was achieved by lymphocytes transduced with immunoGrB containing PE II sequence(280-358 aa). The above data suggest that it is feasible to administrate immunoGrB-secreting autologous lymphocytes to suppress HER-2 positive tumor in vivo.hi summary, this lymphocyte-based antitumour strategy is characteristic of initiation of cell death specially in HER-2 positive tumor. Such is of value, in both theory and clinic, in that immunoGrB potently and selectively kills HER-2 positive tumor cells, and that little immunogenicity makes it possible forlong-term application.