Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

The requirement of two activating proteolytic events by two different tumor-associated proteases may confer the toxic agent with superior cell-type specificity and further attenuate its toxicity to normal tissues. In an elegant work performed by Liu et al [430] , an intermolecular complementation approach was used for the targeting of tumor cells having both MMP and uPA activities which are overproduced by tumor tissues and are implicated in cancer cell growth and metastasis [435] [436] [437] [438] [439] 499, 500] . To this end, the authors exploited the findings that the anthrax LF binding site spans two adjacent monomers of cleaved PA in the oligomeric prepore. Each monomer contains three subsites which play a role in the binding of LF. However, the functional LF binding site is composed of a combination of subsites contributed by two adjacent subunits: subsite I and III form one monomer and subsite II from the neighboring subunit [501, 502] . By mutating LF binding subsite III on an MMP activated PA (PrAg-L1) and subsite II on uPA activated PA (PrAg-U2), assembly of PA heptamer in which every LF binding site contains the inactivating subsite III or subsite II mutation will occur following activating cleavage by either MMP or uPA, respectively. However, adding a mixture of these modified PA proteins to cells that have both MMP and uPA activities would generate two subunits that can randomly assemble into a heptamer in which up to three functional LF binding sites may be generated by intermolecular complementation between the two types of mutated subunits. In order to test the hypothesis that requirement of two tumor-associated activating proteolytic events may attenuate toxicity to normal tissues, an in vivo toxicity assay in mice was performed by intraperitoneal administration of a mixture containing subsite III mutated PrAg-L1 (PrAg-L1-I210A), subsite II mutated PrAg-U2 (PrAg-U2-R200A) and the toxic FP59 chimeric polypeptide. Indeed, results showed decreased toxicity in comparison to administration of a mixture of PrAg-L1, PrAg-U2 and FP59 (where either MMP or uPA proteolytic activity was sufficient to generate a functional LF binding prepore).