The OX-40 receptor (OX-40R) is a member of the tumor necrosis factor receptor (TNF-R) superfamily that is expressed on activated CD4+ T cells. The OX-40R is a costimulatory molecule that induces CD4+ T cell activation when engaged by its ligand (OX-40 L; found on antigen presenting cells). In human and murine tumors, we have shown upregulation of the OX-40R on CD4+ T cells from tumor-infiltrating lymphocytes (TIL) and tumor-draining lymph node cells (TDLNC) but not on systemic CD4+ T cells, such as peripheral blood lymphocytes (PBL) or splenocytes. In order to examine potentially heightened anti-tumor immunity through enhanced costimulation when engaging OX-40R in vivo, we inoculated mice with a murine mammary cancer cell line (SM1) and then treated with a soluble form of the OX-40 L. Mice injected with a lethal inoculum of SM1 cells were given two intraperitoneal injections (days 3 and 7 post-inoculation) of 100 microg soluble OX-40 L. Seven of 28 treated mice survived the lethal tumor inoculum, as compared to one of 28 control mice, demonstrating a significant survival benefit with treatment (p = 0.0136, log rank analysis). Mice that did not develop tumor by day 90 were rechallenged; all remained tumor-free. Mice were also injected with a second mammary tumor line (4T1) and treated with OX-40L:Ig with similar therapeutic results. Activation of OX-40R+ CD4+ T cells during mammary cancer priming stimulated an antitumor immune response resulting in enhanced survival and protective anti-tumor immunity. These results should have practical applications for treatment modalities for patients with breast cancer.

The OX40 ligand (OX40L), a member of TNF superfamily, is a costimulatory molecule involved in T cell activation. It is expressed on antigen presenting cells including dendritic cells (DC) and activated B cells. This molecule has been reported to provide potent costimulation in APC-T cell interactions upon binding to its cognate receptor, OX40 which is expressed by activated T cells. In this study systemic administration of OX40L fusion protein was used in the treatment of established murine subcutaneous colon and breast carcinomas.Intra-peritoneal injection of mOX40L fusion protein significantly inhibited the growth of subcutaneous 3 day established colon (CT26) and breast (4T1) carcinomas which was dose and route dependent. Effective therapy with OX40L required the the presence of tumour for 3 days prior to OX40L, concomitant therapy, given at the same time (day 0) as tumour cells was less effective. Furthermore, therapy with OX40L fusion protein was effective in significantly reducing CT26 experimental lung metastasis. In addition, inhibition of CT26 and 4T1 tumours in response to therapy with OX40L was significantly enhanced by combination treatment with intra-tumour injection of a DISC-HSV vector encoding mGM-CSF. Tumour rejection in response to OX40L therapy was correlated with splenocyte CTL activity against the gp70 CT26 tumour associated antigen. In vivo depletion studies demonstrated the requirement for both CD4+ and CD8+ T cells for effective OX40L therapy.Collectively these results demonstrate the potential role of the OX40L in cancer immunotherapy.3

Previously we showed that addition of purified VP22, a structural protein of herpes simplex virus, to short oligonucleotides (ODN) induced the spontaneous assembly of novel particles incorporating both protein and ODN. These particles were not toxic, entered cells, and resided stably in the cytoplasm. Surprisingly the particles could be activated by light in a regulated synchronous manner to release ODN and protein to the cell cytosol and nuclei. Here we construct a fusion protein containing a short peptide from the proapoptotic BH3 domain family member Bak. The BH3-VP22 protein was recruited into particles that entered cells and remained stable in the cytoplasm without toxicity. Light activation rapidly disrupted the particles, a process captured in living cells by time-lapse microscopy, and this synchronized regulated release resulted in subsequent cell death by apoptosis. In control experiments, particles containing a mutant BH3 peptide, although indistinguishable in cell uptake and regulated release, showed no apoptotic effect. Regulated release of VP22-based particles may find application in mechanistic analysis of apoptotic pathways, in cell-based screening assays both of peptides and of oligonucleotides, or as therapeutic agents incorporating specific additional components.

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