CD40 ligand gene therapy directly induces apoptosis in carcinomas

Abstract

14082 Background: CD40 is a member of the TNFReceptor superfamily expressed on a variety of immune cells. Interaction of CD40 with its ligand (CD40L) plays a key role in orchestrating immune responses. We have shown that CD40 is also expressed on a variety of carcinomas. CD40 ligation in this context can induce cancer cell death. Thus CD40 represents a rational target with direct cytotoxicity and induction of anti-tumour immunity. Methods: We generated an adenovirus encoding CD40L (AdCD40L) and investigated its effect in CD40-expressing carcinoma cells (bladder, ovary, hepatocellular) as follows:(i)comparison of Ad-delivered CD40L with recombinant soluble ligand (rsCD40L);(ii)inhibition of cleavage of CD40L from the cell membrane using metalloproteinase inhibitors (MMPI);(iii)generation of a mutant CD40L resistant to MMP cleavage. Results: (i)CD40 ligation in carcinomas stimulates survival and apoptotic signaling. rsCD40L is only cytotoxic in the presence of protein synthesis inhibition, which blocks survival pathways and allows apoptosis. In contrast, AdCD40L is cytotoxic even in the absence of protein synthesis inhibitor;(ii)AdCD40L results in expression at the cell membrane but this is cleaved from the membrane and detected as soluble ligand in supernatant of infected cells. Cleavage is inhibited by MMPI and this increases apoptosis, suggesting that membrane-bound CD40L is a more potent apoptotic stimulus than soluble ligand;(iii)we identified the cleavage site of CD40L and generated a mutant by deletion of this region and cloned this mCD40L into an adenovirus. AdmCD40L results in membrane expression of CD40L, with no detectable soluble CD40L, confirming its resistance to cleavage. AdmCD40L is significantly more cytotoxic than wild-type AdCD40L. Conclusions: The effect of CD40 ligation depends on the cellular context and the method of CD40L delivery: adenovirus delivery results in membrane-bound expression and this provides a stronger apoptotic stimulus than rsCD40L. Apoptosis is further enhanced by a mutant CD40L that is resistant to cleavage from the cell membrane. Thus AdmCD40L is a novel therapeutic that is a potent inducer of apoptosis. Studies are underway to assess these effects in vivo and to elucidate the contribution of immunostimulatory effects in syngeneic models. No significant financial relationships to disclose.