Mancheño, U. (Uxua)
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- Short-term local expression of a PD-L1 blocking antibody from a self-replicating RNA vector induces potent antitumor responses(2019) Kochan, G. (Grazyna); Mancheño, U. (Uxua); Sanchez-Paulete, A.R. (Alfonso R.); Smerdou, C. (Cristian); Galindo, J. (Javier); Ballesteros-Briones, M.C. (María Cristina); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Casales, E. (Erkuden); Prieto, J. (Jesús); Martisova, E. (Eva); Gorraiz, M. (Marta); Escors, D. (David); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Silva-Pilipich, N.R. (Noelia Romina); Lasarte, J.J. (Juan José)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 s
- Epitope spreading driven by the joint action of CART cells and pharmacological STING stimulation counteracts tumor escape via antigen-loss variants(2021) Rodríguez-García, E. (Estefanía); Vercher-Herráez, E. (Enric); González-Aseguinolaza, G. (Gloria); Conde-Gallastegi, E. (Enrique); Mancheño, U. (Uxua); Uranga-Murillo, I. (Iratxe); Soria-Castellano, M. (Marta); Rodríguez, M.L. (M. Luis); Hommel, M. (Mirja); Alkorta-Aranburu, G. (Gorka); Pardo, J. (Julián); Hervas-Stubbs, S. (Sandra); Suarez-Olmos, J. (Jesús); González-Vaz, J. (Javier); Casares, N. (Noelia); Melero, I. (Ignacio); Elizalde, E. (Edurne); Lasarte, J.J. (Juan José)Background Target antigen (Ag) loss has emerged as a major cause of relapse after chimeric antigen receptor T (CART)-cell therapy. We reasoned that the combination of CART cells, with the consequent tumor debulking and release of Ags, together with an immunomodulatory agent, such as the stimulator of interferon gene ligand (STING-L) 2 ' 3 '-cyclic GMP-AMP (2 ' 3 '-cGAMP), may facilitate the activation of an endogenous response to secondary tumor Ags able to counteract this tumor escape mechanism. Methods Mice bearing B16-derived tumors expressing prostate-specific membrane Ag or gp75 were treated systemically with cognate CART cells followed by intratumoral injections of 2 ' 3 '-cGAMP. We studied the target Ag inmunoediting by CART cells and the effect of the CART/STING-L combination on the control of STING-L-treated and STING-L-non-treated tumors and on the endogenous antitumor T-cell response. The role of Batf3-dependent dendritic cells (DCs), stimulator of interferon gene (STING) signaling and perforin (Perf)-mediated killing in the efficacy of the combination were analyzed. Results Using an immune-competent solid tumor model, we showed that CART cells led to the emergence of tumor cells that lose the target Ag, recreating the cancer immunoediting effect of CART-cell therapy.
- Intensive pharmacological immunosuppression allows for repetitive liver gene transfer with recombinant adenovirus in nonhuman primates(NATURE PUBLISHING GROUP, 2009-12-16) Dubrot, J. (Juan); Benito-Boilos, A. (Alberto); Mancheño, U. (Uxua); Alfaro, C. (Carlos); Collantes, M. (María); Peñuelas-Sanchez, I. (Ivan); Smerdou, C. (Cristian); Unzu, C. (Carmen); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Palazon, A. (Asís); Prieto, J. (Jesús); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Mauleon, I. (Itsaso)Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector.
- Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates(BioMed Central, 2012) Benito-Boilos, A. (Alberto); Mancheño, U. (Uxua); Alfaro, C. (Carlos); Unzu, C. (Carmen); Pety, H. (Harald); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Beattie, S.G. (Stuart G.); Salamanca, E. (Enrique) de; Prieto, J. (Jesús); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Mauleon, I. (Itsaso)BACKGROUND: Adeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this study, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression. METHODS: Three female Macaca fascicularis were intravenously injected with 1x1013 genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5x1012 genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression. RESULTS: Macaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system. CONCLUSIONS: These results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.
- Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice(2011) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Mancheño, U. (Uxua); Alzuguren, P. (Pilar); Hervas-Stubbs, S. (Sandra); Crettaz, J. (Julien); Prieto, J. (Jesús); Medina-Echeverz, J. (José); Hernandez-Alcoceba, R. (Rubén); Gonzalez-Aparicio, M. (Manuela); Mauleon, I. (Itsaso)BACKGROUND AND AIMS: New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. OBJECTIVE: To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. METHODS: A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. RESULTS: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125-4000 μg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in < 25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine.