Jaione Bezunartea

In vitro and in vivo down-regulation of regulatory T cell activity with a peptide inhibitor of TGF-beta1

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-β1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-β1 and TGF-β2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-β may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.

Adjuvant Combination and Antigen Targeting as a Strategy to Induce Polyfunctional and High-Avidity T-Cell Responses against Poorly Immunogenic Tumors

Low antigen expression and an absence of coimmunostimulatory signals may be partly responsible for the low immunogenicity of many tumors. It may be possible to overcome this situation by defining a combination of adjuvants and antigens that can activate a high-avidity antitumor response. Using the poorly immunogenic B16-OVA melanoma cells as tumor model, we tested different combinations of adjuvants and antigens to treat established tumors. In the absence of exogenous antigens, repeated administration of the TLR7 ligand Imiquimod together with anti-CD40 agonistic antibodies activated only innate immunity, which was insufficient to reject intradermal tumors. Administering this adjuvant combination together with OVA as a tumor antigen induced T-cell responses that delayed tumor growth. However, administering a combination of anti-CD40 plus TLR3 and TLR7 ligands, together with antigen targeting to dendritic cells through TLR4, was sufficient to induce tumor rejection in 50% of mice. This response was associated with a greater activation of innate immunity and induction of high-avidity polyfunctional CD8(+) T-cell responses, which each contributed to tumor rejection. This therapy activated T-cell responses not only against OVA, which conferred protection against a rechallenge with B16-OVA cells, but also activated T-cell responses against other melanoma-associated antigens. Our findings support the concept that multiple adjuvant combination and antigen targeting may be a useful immunotherapeutic strategy against poorly immunogenic tumors.

Peptide inhibitors of transforming growth factor-β enhance the efficacy of antitumor immunotherapy

Transforming growth factor‐β (TGF‐β) is a cytokine with potent immunosuppressive effects and is overexpressed in many tumors. Therefore, development of molecules able to inhibit TGF‐β is of paramount importance to improve the efficacy of antitumor immunotherapy. TGF‐β inhibitor peptides P144 and P17 were combined with the administration of adjuvant molecules poly(I:C) and agonistic anti‐CD40 antibodies, and their effect on the growth of E.G7‐OVA established tumors and on antitumor immune response was evaluated. Tumor rejection efficacy of a single administration of adjuvants was enhanced from 15 to 70 % when combined with repeated injections of TGF‐β inhibitor peptides. Simultaneous administration of adjuvants and TGF‐β inhibitor peptides was required for maximal therapeutic efficacy. Although tumor cells produced TGF‐β, it was found that the beneficial effect of peptide administration was mainly due to the inhibition of TGF‐β produced by regulatory CD4+CD25+ T cells rather than by tumor cells. The enhanced antitumor effect was accompanied by a higher activity of dendritic cells, natural killer cells and tumor antigen‐specific T cells, as well as by a decrease in the number of myeloid‐derived suppressor cells. In conclusion, administration of peptide inhibitors of TGF‐β in therapeutic vaccination enhances the efficacy of immunotherapy by increasing antitumor immune responses. These peptide inhibitors may have important applications for current immunotherapeutic strategies.

Immunotherapeutic synergy between anti-CD137 mAb and intratumoral administration of a cytopathic Semliki Forest virus encoding IL-12.

Intratumoral injection of Semliki Forest virus encoding interleukin-12 (SFV-IL-12) combines acute expression of IL-12 and stressful apoptosis of infected malignant cells. Agonist antibodies directed to costimulatory receptor CD137 (4-1BB) strongly amplify pre-existing cellular immune responses toward weak tumor antigens. In this study, we provide evidence for powerful synergistic effects of a combined strategy consisting of intratumoral injection of SFV-IL-12 and systemic delivery of agonist anti-CD137 monoclonal antibodies (mAbs), which was substantiated against poorly immunogenic B16 melanomas (B16-OVA and B16.F10) and TC-1 lung carcinomas. Effector CD8(β)(+) T cells were sufficient to mediate complete tumor eradications. Accordingly, there was an intensely synergistic in vivo enhancement of cytotoxic T lymphocytes (CTL)-mediated immunity against the tumor antigens OVA and tyrosine-related protein-2 (TRP-2). This train of phenomena led to long-lasting tumor-specific immunity against rechallenge, attained transient control of the progression of concomitant tumor lesions that were not directly treated with SFV-IL-12 and caused autoimmune vitiligo. Importantly, we found that SFV-IL-12 intratumoral injection induces bright expression of CD137 on most tumor-infiltrating CD8(+) T lymphocytes, thereby providing more abundant targets for the action of the agonist antibody. This efficacious combinatorial immunotherapy strategy offers feasibility for clinical translation since anti-CD137 mAbs are already undergoing clinical trials and development of clinical-grade SFV-IL-12 vectors is in progress.

Eradication of Liver-Implanted Tumors by Semliki Forest Virus Expressing IL-12 Requires Efficient Long-Term Immune Responses

Semliki Forest virus vectors expressing IL-12 (SFV–IL-12) were shown to induce potent antitumor responses against s.c. MC38 colon adenocarcinomas in immunocompetent mice. However, when MC38 tumors were implanted in liver, where colon tumors usually metastasize, SFV–IL-12 efficacy was significantly reduced. We reasoned that characterization of immune responses against intrahepatic tumors in responder and nonresponder animals could provide useful information for designing more potent antitumor strategies. Remarkably, SFV–IL-12 induced a high percentage of circulating tumor-specific CD8 T cells in all treated animals. Depletion studies showed that these cells were essential for SFV–IL-12 antitumor activity. However, in comparison with nonresponders, tumor-specific cells from responder mice acquired an effector-like phenotype significantly earlier, were recruited more efficiently to the liver, and, importantly, persisted for a longer period of time. All treated mice had high levels of functional specific CD8 T cells at 8 d posttreatment reflected by both in vivo killing and IFN-γ–production assays, but responder animals showed a more avid and persistent IFN-γ response. Interestingly, differences in immune responses between responders and nonresponders seemed to correlate with the immune status of the animals before treatment and were not due to the treatment itself. Mice that rejected tumors were protected against tumor rechallenge, indicating that sustained memory responses are required for an efficacious therapy. Interestingly, tumor-specific CD8 T cells of responder animals showed upregulation of IL-15Rα expression compared with nonresponders. These results suggest that SFV–IL-12 therapy could benefit from the use of strategies that could either upregulate IL-15Rα expression or activate this receptor.

Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions.

We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under normal and oxidative stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5 days after treatment. Finally, none of the drugs affected migration. In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.

Capsid-deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane

Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.

Recent Patents on Alphavirus Protein Expression and Vector Production

Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

Helper cell-independent antitumor activity of potent CD8(+) T cell epitope peptide vaccines is dependent upon CD40L.

Peptide vaccines derived from CD8+ T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8+ T-cell responses, even in the absence of CD4+ T-cell epitopes or dendritic cell (DC)-activating adjuvants. However, the mechanisms underlying the clinical activity of these potent peptides are poorly understood. Using CT26 and ovalbumin-expressing B16 murine allograft tumor models, we found that the antitumor effect of helper cell-independent CD8 T-cell peptide vaccines is inhibited by the blockade of CD40 ligand (CD40L) in vivo. Furthermore, in vitro stimulation with antigenic peptides of cells derived from immunized mice induced the expression of CD40L on the surface of CD8+ T cells and fostered DC maturation, an effect that was partially inhibited by CD40L-blocking antibodies. Interestingly, CD40L blockade also inhibited CD8+ T-cell responses, even in the presence of fully mature DCs, suggesting a role for CD40L not only in promoting DC maturation but also in mediating CD8+ T-cell co-stimulation. Importantly, these potent peptides share features with bona fide CD4 epitopes, since they foster responses against less immunogenic CD8+ T-cell epitopes in a CD40L-dependent manner. The analysis of peptides used for the vaccination of cancer patients in clinical trials showed that these peptides also induce the expression of CD40L on the surface of CD8+ T cells. Taken together, these results suggest that CD40L expression induced by potent CD8+ T-cell epitopes can activate antitumor CD8+ T-cell responses, potentially amplifying the immunological responses to less immunogenic CD8+ T-cell epitopes and bypassing the requirement for CD4+ helper T cells in vaccination protocols.

Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery

Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng) PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.