Minit-Livin Conjugated to Targeted Nanoparticles Inhibits Tumorigenicity

We found that Livin, an Inhibitor of Apoptosis Protein, is specifically cleaved to produce a truncated Livin protein (tLivin) and demonstrated the paradoxical pro-apoptotic activity of tLivin. mini-tLivin (mtLivin) is a 70 aa derivative of tLivin that remarkably, exhibits a pro-apoptotic activity as potent as tLivin. Our findings regarding tLivin and mtLivin suggest unique therapeutic avenue against cancer. To improve the delivery and efficacy of mtLivin for treatment we conjugated mtLivin-to nanoparticles (NPs) of poly (lactide-co-glycolide) (PLGA) and targeted the nano-delivery system to diffuse large B-cell lymphoma (DLBCL) using CD40L. Delivery of mtLivin with nanoparticles should enhance stability, provide prolonged bioavailability, intracellular uptake and efficacy.

We generated bifunctional mtLivin-CD40L-NPs to target mtLivin-NPs to DLBCL cells. Various cell lines were used to evaluate the biological activity of free and conjugated (targeted or not) mtLivin including 293T, 721.221 B cells, L428 Hodgkin's lymphoma, OCI-LY19 DLBCL cells and OCI-LY19- GLuc cells that constitutively express a gaussia luciferase reporter.

Targeted, bifunctional mtLivin-CD40L-NPs elicited significant cell death of DLBCL cells while monofunctional mtLivin-NPs had a lower effect. mtLivin-NPs may induce cell death of some cell lines without targeting (e.g. 293T) yet targeting is required to induce marked cell death of DLBCL cells.

To best mimic lymphoma disease in humans, a disseminated lymphoma model was used to evaluate the antitumor effect of targeted mtLivin treatment. NOD/SCID mice were injected with OCI-LY19- GLuc cells or into the tail vein. Gaussia luciferase reporter allows IVIS imaging to monitor tumor size and location in mice. Mice were treated approximately once a week with various mtLivin-NPs formulations. Luciferase signal was observed as early as day 2 after tumor cells injection.

Immunohistochemistry for anti-CD20, a human B-cell marker, showed infiltration of OCI-ly19-Gluc cells in spleen and brain close to tumor cells injection (Day 2). With time, lymphoma infiltrations were detected in brain, bone marrow, lungs, spleen and liver. All control mice exhibited paralysis and did not survive beyond 28 days. In contrast, 37.5% of animals receiving mtLivin-NPs and 71.4 % of animals receiving mtLivin-CD40L-NPs achieved complete pathological tumor response and survived significantly longer. All treatments in this study were well-tolerated.

In terms of mechanism, we show high caspase-3 activity in tumors from mice that were treated with mtLivin-CD40L-NPs demonstrating the ability of the nanoparticles to target the tumors and to induce apoptotic tumor cell death. The resistance of tumor cells to drug-induced apoptosis is a major cause of cancer treatment failure. We designed mtLitvin targeted nanoparticles constituting novel personalized therapeutic approach for resistant cancer.