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Apoptotic agents

The serine-dependent and aspartate-specific protease granzyme B is of particular interest. Granzyme B is a component of cellular immune defense which, upon activation of cytotoxic T cells (CTL) or natural killer cells (NK), is secreted from the cytotoxic granules of these cells. Upon the perforin-dependent translocation of granzyme B into the cytoplasm of attacked cells, a proteolytic cascade is initiated which ends in the apoptosis of the target cell. The exact function of the perforin secreted along with granzyme B is still being discussed currently, but it is not capable of inducing apoptosis alone.

In the cell membrane, perforin aggregates into 12-18mers and thereby forms pores of 15-18 nm. Initially, it was considered that granzyme B gets into the cytoplasm of the target cells through these pores. However, the 32 kDa protein granzyme B is too large for such a passage. It is more probable to assume that, after granzyme B has bound to perforin and this complex is successively internalized, perforin supports the endosomal release of granzyme B.

In recent years, various proteins could be identified which are activated by GB-mediated cleavage are directly related to apoptosis. Thus, the GB-caused proteolytic activation of various procaspases, especially 3 and 8, could be documented in vitro; these are counted with the central proteases in apoptosis. Further cytotoxic activities are displayed by granzyme B in the nucleus. After having intruded the cytoplasms of the target cell, granzyme B is relatively quickly translocated into the nucleus in a caspase-dependent way. There, granzyme B is capable, for example, of cutting nuclear matrix antigen and poly(ADP-ribose) polymerase. A quick apoptosis could be observed in cells after granzyme B accumulated in the nucleus. More recent data prove the initiation of apoptosis through the direct proteolytic cleavage of Bid, a member of the Bcl-2 family having only one BH3 domain. After cleavage, the truncated form tBid becomes embedded in the mitochondrial membrane and depolarizes it. This induces the release of cytochrome c and an apoptosis-inducing factor from the mitochondria into the cytoplasm, which critically accelerated cell death. Further caspase-independent toxic properties of granzyme B could be described, the underlying mechanism still being uncleared. We generated new human immunotoxins granzyme B to recombinant antibody fragments (WO01/80880).