Nuestros investigadores

Cristian Smerdou Picazo

Publicaciones científicas más recientes (desde 2010)

Autores: Ballesteros-Briones, M. C. ; Martisová, Eva; et al.
ISSN 1525-0016  Vol. 27  Nº 11  2019  págs. 1892 - 1905
Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFVaPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.
Autores: Weber, N. D.; Odriozola, L.; et al.
ISSN 1525-0016  Vol. 27  Nº 4  2019  págs. 203 - 203
Autores: Martisová, Eva; Ballesteros-Briones, M. C.; et al.
ISSN 1043-0342  Vol. 30  Nº 11  2019  págs. A57 - A57
Autores: Ballesteros-Briones, M. C.; Martisová, Eva; et al.
ISSN 1043-0342  Vol. 30  Nº 11  2019  págs. A16 - A16
Autores: Ballesteros-Briones, M. C.; Martisová, Eva; et al.
ISSN 1525-0016  Vol. 27  Nº 4  2019  págs. 268 - 268
Autores: Sanchez-Paulete, A. R. ; Quetglas, J. I.; et al.
ISSN 0008-5472  Vol. 78  Nº 23  2018  págs. 6643 - 6654
Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38-and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8 beta T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNARdeficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltratingCD8T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. Significance: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates crosspriming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion. (C) 2018 AACR.
Autores: Gato-Cañas, M. ; Zuazo, M.; Arasanz, H. ; et al.
ISSN 2211-1247  Vol. 20  Nº 8  2017  págs. 1818 - 1829
PDL1 blockade produces remarkable clinical responses, thought to occur by T cell reactivation through prevention of PDL1-PD1 T cell inhibitory interactions. Here, we find that PDL1 cell-intrinsic signaling protects cancer cells from interferon (IFN) cytotoxicity and accelerates tumor progression. PDL1 inhibited IFN signal transduction through a conserved class of sequence motifs that mediate crosstalk with IFN signaling. Abrogation of PDL1 expression or antibody-mediated PDL1 blockade strongly sensitized cancer cells to IFN cytotoxicity through a STAT3/caspase-7-dependent pathway. Moreover, somatic mutations found in human carcinomas within these PDL1 sequence motifs disrupted motif regulation, resulting in PDL1 molecules with enhanced protective activities from type I and type II IFN cytotoxicity. Overall, our results reveal a mode of action of PDL1 in cancer cells as a first line of defense against IFN cytotoxicity.
Autores: Rodriguez-Madoz, Juan Roberto; Alfaro M; Prieto, Jesús María; et al.
ISSN 1043-0342  Vol. 25  Nº 2  2014  págs. 132 - 143
Interleukin-12 (IL-12) is an immunostimulatory cytokine that has shown strong antitumor effects in animal models of liver cancer. In order to overcome the severe toxicity associated with its systemic administration, we had previously tested different strategies based on IL-12 gene transfer to tumor cells or to the surrounding liver tissue. We obtained promising results both with a recombinant Semliki Forest virus (SFV) vector expressing high levels of IL-12 (SFV-IL-12) after intratumoral injection and with a plasmid vector [pTonL2(T)-mIL12] that allows liver-specific and inducible IL-12 expression. The aim of the present study was to compare the antitumor responses induced by both systems in a clinically relevant animal model of hepatocellular carcinoma (HCC) developed in L-PK/c-myc transgenic mice. These animals overexpress the c-myc oncogene in their livers, giving rise to spontaneous hepatic tumors with latency, histopathology, and genetic characteristics similar to human HCCs. We observed that intratumoral inoculation of SFV-IL-12 induced growth arrest in most tumors, providing 100% survival rate, in contrast to no survival in control animals. Similar results were obtained with hydrodynamic injection of pTonL2(T)-mIL12 after long-term induction of IL-12 expression in the liver. However, tumor arrest was less evident in plasmid-treated mice and the survival rate was slightly lower, despite higher and more sustained levels of IL-12 and IFN-¿ in serum. The fact that SFV-IL-12 was able to induce both apoptosis and a type-I IFN response specifically in the tumor could explain why short-term IL-12 expression from this vector was sufficient to mediate an antitumoral response comparable with long-term IL-12 expression driven by pTonL2(T)-mIL12. Since SFV-IL-12 could reduce the possible toxicity associated with long-term IL-12 expression, we believe that this vector could have a potential application for HCC gene therapy.
Autores: Melero, Ignacio Javier; Rodriguez-Madoz, Juan Roberto; et al.
ISSN 0008-5472  Vol. 75  Nº 3  2014  págs. 497 - 507
Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFN beta transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFN beta signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expand
Autores: Bezunartea J; Rodriguez-Madoz, Juan Roberto; et al.
ISSN 1420-682X  Vol. 71  Nº 23  2014  págs. 4637 - 4651
We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.
Autores: Sandra Hervas-Stubbs; Smerdou, Cristian;
ISSN 2162-4011  Vol. 2  Nº 6  2013  págs. e24499
Do cancer patients responding to immunotherapy have immunological profiles that influence the therapeutic outcome, or do they develop efficient antitumor responses only upon immunotherapy? We came across this "chicken or the egg" dilemma when treating secondary liver tumors with Semliki Forest viruses expressing interleukin-12. In our system, the "egg," that is, the pre-treatment immunological profile, seemed to make the difference. The properties of an effective antitumor response were also defined.
Autores: Ansorena, Eduardo; et al.
ISSN 0378-5173  Vol. 440  Nº 1  2013  págs. 19-26
Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes. (C) 2012 Elsevier B.V. All rights reserved.
Autores: Rodriguez-Madoz, Juan Roberto; Bezunartea, J.; et al.
ISSN 0022-1767  Vol. 190  Nº 6  2013  págs. 2994 - 3004
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-¿-production assays, but responder animals showed a more avid and persistent IFN-¿ 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-15R¿ expression compared with nonresponders. These results suggest that SFV-IL-12 therapy could benefit from the use of strategies that could either upregulate IL-15R¿ expression or activate this receptor.
Autores: Quetglas, José Ignacio; Baraibar, I.; et al.
Revista: Gene therapy (Basingstoke) (print)
ISSN 0969-7128  Vol. 19  Nº 3  2012  págs. 271 - 278
Autores: Quetglas, José Ignacio; John, L.B.; Kershaw, M.H.; et al.
ISSN 2162-4011  Vol. 1  Nº 8  2012  págs. 1345 - 1355
Malignant cells are susceptible to viral infection and consequent cell death. Virus-induced cell death is endowed with features that are known to stimulate innate and adaptive immune responses. Thus danger signals emitted by cells succumbing to viral infection as well as viral nucleic acids are detected by specific receptors, and tumor cell antigens can be routed to professional antigen-presenting cells. The anticancer immune response triggered by viral infection is frequently insufficient to eradicate malignancy but may be further amplified. For this purpose, transgenes encoding cytokines as co-stimulatory molecules can be genetically engineered into viral vectors. Alternatively, or in addition, it is possible to use monoclonal antibodies that either block inhibitory receptors of immune effector cells, or act as agonists for co-stimulatory receptors. Combined strategies are based on the ignition of a local immune response at the malignant site plus systemic immune boosting. We have recently reported examples of this approach involving the Vaccinia virus or Semliki Forest virus, interleukin-12 and anti-CD137 monoclonal antibodies.
Autores: Bezunartea, Jaione; et al.
ISSN 1525-0016  Vol. 20  Nº 9  2012  págs. 1664 - 1675
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(beta)(+) 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.
Autores: Quetglas, José Ignacio; Bezunartea, Jaione; et al.
ISSN 1872-2083  Vol. 5  Nº 3  2011  págs. 212 - 226
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.
Autores: Smerdou, Cristian; Ochoa, María del Carmen; Quetglas, José Ignacio; et al.
Revista: Molecular Therapy
ISSN 1525-0016  Nº 18  2010  págs. 456 - 459
Autores: Quetglas, José Ignacio; et al.
ISSN 1871-6784  Vol. 27  Nº 2  2010  págs. 138 - 148
Autores: Quetglas, José Ignacio; et al.
Revista: Virus Research
ISSN 0168-1702  Vol. 153  Nº 2  2010  págs. 179 - 196
Autores: Fontanellas, Antonio; Sandra Hervas-Stubbs; et al.
Revista: Molecular Therapy
ISSN 1525-0016  Vol. 18   Nº 4  2010  págs. 754 - 765
Autores: Smerdou, Cristian; Menne, S.; Hernández, Rubén; et al.
Revista: Current Opinion in Investigational Drugs
ISSN 1472-4472  Vol. 11  Nº 12  2010  págs. 1368 - 1377