Rodney A. Prell

Engagement of the OX-40 Receptor In Vivo Enhances Antitumor Immunity

The OX-40 receptor (OX-40R), a member of the TNFR family, is primarily expressed on activated CD4+ T lymphocytes. Engagement of the OX-40R, with either OX-40 ligand (OX-40L) or an Ab agonist, delivers a strong costimulatory signal to effector T cells. OX-40R+ T cells isolated from inflammatory lesions in the CNS of animals with experimental autoimmune encephalomyelitis are the cells that respond to autoantigen (myelin basic protein) in vivo. We identified OX-40R+ T cells within primary tumors and tumor-invaded lymph nodes of patients with cancer and hypothesized that they are the tumor-Ag-specific T cells. Therefore, we investigated whether engagement of the OX-40R in vivo during tumor priming would enhance a tumor-specific T cell response. Injection of OX-40L:Ig or anti-OX-40R in vivo during tumor priming resulted in a significant improvement in the percentage of tumor-free survivors (20–55%) in four different murine tumors derived from four separate tissues. This anti-OX-40R effect was dose dependent and accentuated tumor-specific T cell memory. The data suggest that engagement of the OX-40R in vivo augments tumor-specific priming by stimulating/expanding the natural repertoire of the host’s tumor-specific CD4+ T cells. The identification of OX-40R+ T cells clustered around human tumor cells in vivo suggests that engagement of the OX-40R may be a practical approach for expanding tumor-reactive T cells and thereby a method to improve tumor immunotherapy in patients with cancer.

Danger and OX40 Receptor Signaling Synergize to Enhance Memory T Cell Survival by Inhibiting Peripheral Deletion

This report defines a cell surface receptor (OX40) expressed on effector CD4 T cells, which when engaged in conjunction with a danger signal, rescues Ag-stimulated effector cells from activation-induced cell death in vivo. Specifically, three signals were necessary to promote optimal generation of long-lived CD4 T cell memory in vivo: Ag, a danger signal (LPS), and OX40 engagement. Mice treated with Ag or superantigen (SAg) alone produced very few SAg-specific T cells. OX40 ligation or LPS stimulation, enhanced SAg-driven clonal expansion and the survival of responding T cells. However, when SAg was administered with a danger signal at the time of OX40 ligation, a synergistic effect was observed which led to a 60-fold increase in the number of long-lived, Ag-specific CD4 memory T cells. These data lay the foundation for the provision of increased numbers of memory T cells which should enhance the efficacy of vaccine strategies for infectious diseases, or cancer, while also providing a potential target (OX40) to limit the number of auto-Ag-specific memory T cells in autoimmune disease.

Vascular Endothelial Growth Factor Blockade Reduces Intratumoral Regulatory T Cells and Enhances the Efficacy of a GM-CSF–Secreting Cancer Immunotherapy

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)–secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. Experimental Design: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a soluble VEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory T cells were quantified by their CD4+CD25hi and CD4+FoxP3+ phenotype. Results: The present study established that GM-CSF–secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4+ and CD8+ tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory T cells in the tumor, suggesting a novel mechanism of VEGF-mediated immune suppression at the tumor site. Conclusion: GM-CSF–secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.

Engagement of OX40 Enhances Antigen-Specific CD4+ T Cell Mobilization/Memory Development and Humoral Immunity: Comparison of αOX-40 with αCTLA-4

Increasing the long-term survival of memory T cells after immunization is key to a successful vaccine. In the past, the generation of large numbers of memory T cells in vivo has been difficult because Ag-stimulated T cells are susceptible to activation-induced cell death. Previously, we reported that OX40 engagement resulted in a 60-fold increase in the number of Ag-specific CD4+ memory T cells that persisted 60 days postimmunization. In this report, we used the D011.10 adoptive transfer model to examine the kinetics of Ag-specific T cell entry into the peripheral blood, the optimal route of administration of Ag and αOX40, and the Ag-specific Ab response after immunization with soluble OVA and αOX40. Finally, we compared the adjuvant properties of αOX40 to those of αCTLA-4. Engagement of OX-40 in vivo was most effective when the Ag was administered s.c. Time course studies revealed that it was crucial for αOX40 to be delivered within 24–48 h after Ag exposure. Examination of anti-OVA Ab titers revealed a 10-fold increase in mice that received αOX40 compared with mice that received OVA alone. Both αOX40 and αCTLA-4 increased the percentage of OVA-specific CD4+ T cells early after immunization (day 4), but αOX40-treated mice had much higher percentages of OVA-specific memory CD4+ T cells from days 11 to 29. These studies demonstrate that OX40 engagement early after immunization with soluble Ag enhances long-term T cell and humoral immunity in a manner distinct from that provided by blocking CTLA-4.

OX40-Mediated Memory T Cell Generation Is TNF Receptor-Associated Factor 2 Dependent

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.

Efficacy of GM-CSF-producing Tumor Vaccine after Docetaxel Chemotherapy in Mice Bearing Established Lewis Lung Carcinoma

In this report, we evaluated the efficacy of a GM-CSF-producing tumor vaccine given before and after docetaxel in mice bearing established lung tumors. Mice bearing established 3LL tumors were treated with docetaxel and tumor vaccines transduced with either control or GM-CSF adenoviral vectors. Docetaxel (5–20u2009mg/kg) treatment alone had only a minimal effect on growth of established 3LL tumors in vivo, although docetaxel was cytotoxic to 3LL cells in vitro. When mice bearing established 3LL tumors were pretreated with docetaxel followed by vaccination with irradiated GM-CSF- transduced 3LL tumor cells, significant tumor regression and prolonged survival were observed compared with chemotherapy alone. Delaying docetaxel treatment until after tumor vaccination abrogated the vaccines anti-tumor effects. Mice that survived treatment were able to resist a lethal rechallenge of 3LL tumor cells. Memory CTL specific for an epitope (MUT-1) derived from 3LL were detected in surviving mice. Docetaxel induced a mild lymphodepletion in mice, both CD4 and CD8 subsets were reduced in LN and spleens. Interestingly, docetaxel also diminished the number of memory CD8+ T cells (CD122+) and possible CD4+CD25+Foxp3+ natural Treg cells. Docetaxel treatment did not affect antigen-driven proliferation of naive T cells but significantly promoted survival of activated T cells. Thus, augmentation of vaccine induced antitumor immunity in docetaxel-treated mice primarily due to the enhanced survival of antigen-experienced T cells.

The generation of T cell memory: a review describing the molecular and cellular events following OX40 (CD134) engagement

OX40 (CD134), a membrane‐bound member of the tumor necrosis factor‐receptor superfamily, is expressed primarily on activated CD4+ T cells. Following engagement on the cell surface, OX40 delivers a costimulatory signal that leads to potent, proinflammatory effects. Engagement of OX40 during antigen (Ag)‐specific stimulation of T cells leads to increased production of memory T cells, increased migration of Ag‐specific T cells, enhanced cytokine production by effector T cells, and the ability to break peripheral T cell tolerance in vivo. Therefore, OX40 engagement in vivo could have important ramifications for the enhancement of vaccine strategies and inhibition of unwanted inflammation. This review summarizes the molecular and cellular events that occur following OX40 engagement during Ag‐specific T cell activation.

The anti-tumor efficacy of a GM-CSF-secreting tumor cell vaccine is not inhibited by docetaxel administration

Docetaxel has demonstrated therapeutic efficacy against breast, prostate, and ovarian cancer and other solid tumors. The tumoricidal activity of docetaxel is mainly attributed to its ability to block microtubule depolymerization, thus inducing G2-M arrest and apoptosis. Mounting evidence indicates that docetaxel also possesses immunomodulatory activity such as augmenting macrophage and lymphokine activated killer activity and inducing pro-inflammatory cytokines, suggesting that docetaxel may be a good chemotherapeutic agent to combine with cancer immunotherapies, assuming that it does not inhibit the vaccine-induced immune response. The anti-tumor activity of the combination of docetaxel and a GM-CSF-secreting B16F10 tumor cell vaccine (B16.GM) was evaluated in the murine B16 melanoma model. Dose levels of docetaxel and the B16.GM vaccine known to be ineffective when used as single agents were selected. Three iv treatments of 6xa0mg/kg docetaxel per injection given on days 5, 9, and 13 after tumor challenge or a single vaccination with 2–3×106 B16.GM cells on day 3 were ineffective at inhibiting tumor growth when used as single agents [median survival time (MST)=24xa0days in both treatment groups and in control animals]. However, combination of docetaxel and B16.GM vaccine significantly delayed tumor growth, increasing MST to 45xa0days. A similar improvement in anti-tumor efficacy was observed using multiple treatment cycles of the B16.GM vaccine/docetaxel combination. Administration of docetaxel every 4xa0days between bi-weekly B16.GM vaccinations increased the median survival of tumor-bearing mice from 31 to 52xa0days compared to multiple B16.GM vaccinations alone. In summary, these data demonstrate that rather than inhibiting the anti-tumor effects of a GM-CSF-secreting tumor cell vaccine, docetaxel combined with a whole cell vaccine significantly inhibits tumor growth, increases survival time and does not impede T-cell activation in the murine B16F10 melanoma tumor model. GM-secreting tumor cell vaccines in combination with docetaxel may represent a new strategy for combining chemo and immunotherapy for cancer.

Activation of immature hepatic NK cells as immunotherapy for liver metastatic disease.

NK cells can identify and eliminate emerging tumors due to altered expression of activating and inhibitory ligands on aberrant cells, a process that is greatly enhanced following NK cell activation. As a principal site of both tumor metastases and immature NK cells, the liver represents a unique anatomic location in which activation of the innate immune system could provide substantial therapeutic benefit. We describe here the NK cell-dependent destruction of a primary hepatic tumor following infection with an attenuated intracellular bacterium derived from Listeria monocytogenes. NK cell-mediated immunity correlated with the ordered migration and maturation of NK cells within the liver. Cytolytic activity was partially dependent on NKG2D-mediated tumor cell recognition, but surprisingly was still effective in the absence of type I IFN. Significantly, NK cell-mediated destruction of a primary hepatic tumor in infected mice led to long-lived CD4- and CD8 T cell-dependent tumor-specific adaptive immunity. These findings establish that activation and differentiation of immature NK cells using complex microbial stimuli can elicit potent anti-tumor activity within the liver, promote cross-presentation of tumor-derived Ags leading to long-lived systemic anti-tumor immunity, and suggests a paradigm for clinical intervention of liver metastatic carcinoma.

Administration of IFN-α Enhances the Efficacy of a Granulocyte Macrophage Colony Stimulating Factor–Secreting Tumor Cell Vaccine

IFN-alpha is approved for the treatment of multiple cancers. Its pleiotropic properties include inhibition of proliferation and angiogenesis and induction of apoptosis. Type I IFNs also exert immunomodulatory effects, which make it an appropriate candidate to combine with cancer vaccines. The studies reported herein show that 50% of mice reject established B16 tumors following treatment with the combination of a granulocyte macrophage colony-stimulating factor-secreting tumor cell vaccine (B16.GM) and subclinical doses of recombinant murine IFN-alpha delivered at the vaccine site. Similarly, 80% of mice treated with the combination reject established B16 tumors when recombinant murine IFN-alpha is given at the challenge site, suggesting that in the latter case its antiproliferative, proapoptotic, and antiangiogenic properties may be involved in controlling tumor growth. In contrast, fewer than 10% of mice reject the tumors when either one is used as a monotherapy. Furthermore, a 30-fold increase in the frequency of melanoma-associated antigen (Trp-2 and gp100) specific T cells was observed in mice treated with the combination when compared with unvaccinated controls. These data show that IFN-alpha combined with a granulocyte macrophage colony-stimulating factor-secreting tumor cell vaccine significantly enhances vaccine potency and may represent a potential new approach for tumor immunotherapy.