Etxeberria, I. (Iñaki)
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- Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15(Taylor & Francis, 2018) Berraondo, P. (Pedro); Gomar, C. (Celia); Etxeberria, I. (Iñaki); Perez-Ruiz, E. (Elisabeth); Rodriguez, I. (Inmaculada); Mayer, J.P. (Jan Peter); Garasa, S. (Saray); Inoges, S. (Susana); Lopez, A. (Ascensión); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Vasquez, M. (Marcos); Wirtz, P. (Peter); Minute, L. (Luna)Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8+ T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8+ T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo. The EGFR+ human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2-/-γc-/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1-/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens.
- Cytokines in clinical cancer immunotherapy(Springer Science and Business Media LLC, 2019) Berraondo, P. (Pedro); Etxeberria, I. (Iñaki); Perez-Gracia, J.L. (Jose Luis); Rodriguez-Ruiz, M.E. (María Esperanza); Aznar, M.A. (María Ángela); Castañon, E. (Eduardo); Ponz-Sarvise, M. (Mariano); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Fernandez-Sanmamed, M. (Miguel)Cytokines are soluble proteins that mediate cell-to-cell communication. Based on the discovery of the potent anti-tumour activities of several pro-inflammatory cytokines in animal models, clinical research led to the approval of recombinant interferon-alpha and interleukin-2 for the treatment of several malignancies, even if efficacy was only modest. These early milestones in immunotherapy have been followed by the recent addition to clinical practice of antibodies that inhibit immune checkpoints, as well as chimeric antigen receptor T cells. A renewed interest in the anti-tumour properties of cytokines has led to an exponential increase in the number of clinical trials that explore the safety and efficacy of cytokine-based drugs, not only as single agents, but also in combination with other immunomodulatory drugs. These second-generation drugs under clinical development include known molecules with novel mechanisms of action, new targets, and fusion proteins that increase half-life and target cytokine activity to the tumour microenvironment or to the desired effector immune cells. In addition, the detrimental activity of immunosuppressive cytokines can be blocked by antagonistic antibodies, small molecules, cytokine traps or siRNAs. In this review, we provide an overview of the novel trends in the cytokine immunotherapy field that are yielding therapeutic agents for clinical trials.
- Immunotherapeutic effects of intratumoral nanoplexed poly I:C(BMC, 2019) Planelles, M. (María); Etxeberria, I. (Iñaki); Perez-Gracia, J.L. (Jose Luis); Rodriguez, I. (Inmaculada); Bolaños, E. (Elixabet); Rodriguez-Ruiz, M.E. (María Esperanza); Aznar, M.A. (María Ángela); Marquez-Rodas, I. (Iván); Garasa, S. (Saray); Quintero, M. (Marisol); Lopez-Casas, P. (Pedro); Molina, C. (Carmeen); Melero, I. (Ignacio); Perez, G. (Guiomar); Perez-Olivares, M. (Mercedes); Teijeira, A. (Álvaro)Poly I:C is a powerful immune adjuvant as a result of its agonist activities on TLR-3, MDA5 and RIG-I. BO-112 is a nanoplexed formulation of Poly I:C complexed with polyethylenimine that causes tumor cell apoptosis showing immunogenic cell death features and which upon intratumoral release results in more prominent tumor infiltration by T lymphocytes. Intratumoral treatment with BO-112 of subcutaneous tumors derived from MC38, 4 T1 and B16- F10 leads to remarkable local disease control dependent on type-1 interferon and gamma-interferon. Some degree of control of non-injected tumor lesions following BO-112 intratumoral treatment was found in mice bearing bilateral B16-OVA melanomas, an activity which was enhanced with co-treatment with systemic anti-CD137 and anti-PD-L1 mAbs. More abundant CD8+ T lymphocytes were found in B16-OVA tumor-draining lymph nodes and in the tumor microenvironment following intratumoral BO-112 treatment, with enhanced numbers of tumor antigen-specific cytotoxic T lymphocytes. Genome-wide transcriptome analyses of injected tumor lesions were consistent with a marked upregulation of the type-I interferon pathway. Inspired by these data, intratumorally delivered BO-112 is being tested in cancer patients (NCT02828098).
- Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma(2023) Glez-Vaz, J. (Javier); Berraondo, P. (Pedro); Cirella, A. (Assunta); Etxeberria, I. (Iñaki); Álvarez-Rodríguez, M. (Maite); Sangro, B. (Bruno); Azpilicueta, A. (Arantza); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Andrea, C.E. (Carlos Eduardo) de; Argemí, J. (Josepmaria); Olivera, I. (Irene); Luri-Rey, C. (Carlos); Sánchez-Gregorio, S. (Sandra); Teijeira, A. (Álvaro)Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular car-cinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co -expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addi-tion of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 reg-imens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.
- Repurposing the yellow fever vaccine for intratumoral immunotherapy(EMBO, 2020) Berraondo, P. (Pedro); Ríus-Rocabert, S. (Sergio); Etxeberria, I. (Iñaki); Azpilikueta, A. (Arantza); Sanchez-Paulete, A.R. (Alfonso R.); Rodriguez, I. (Inmaculada); Nistal-Villan, E. (Estanislao); Aznar, M.A. (María Ángela); Cordeiro-Minute, L. R (Luna Ridan); Garasa, S. (Saray); Molina, C. (Carmeen); Melero, I. (Ignacio); Álvarez, M. (Maite); Teijeira, A. (Álvaro)Live 17D is widely used as a prophylactic vaccine strain for yellow fever virus that induces potent neutralizing humoral and cellular immunity against the wild-type pathogen. 17D replicates and kills mouse and human tumor cell lines but not non-transformed human cells. Intratumoral injections with viable 17D markedly delay transplanted tumor progression in a CD8 T-cell-dependent manner. In mice bearing bilateral tumors in which only one is intratumorally injected, contralateral therapeutic effects are observed consistent with more prominent CD8 T-cell infiltrates and a treatment-related reduction of Tregs. Additive efficacy effects were observed upon co-treatment with intratumoral 17D and systemic anti-CD137 and anti-PD-1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized with 17D, intratumoral 17D treatment achieved better local and distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8 T-cell infiltration in the treated tumor. The repurposed use of a GMP-grade vaccine to be given via the intratumoral route in prevaccinated patients constitutes a clinically feasible and safe immunotherapy approach.
- Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death(2020) Berraondo, P. (Pedro); Etxeberria, I. (Iñaki); Azpilikueta, A. (Arantza); Miguéliz-Basterra, I. (Itziar); Otano, I. (Itziar); Molina, C. (Carmeen); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Andrea, C.E. (Carlos Eduardo) de; Álvarez, M. (Maite); Minute, L. (Luna); Fernandez-Sanmamed, M. (Miguel); Teijeira, A. (Álvaro)Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinicalgrade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies.
- Intercellular adhesion molecule-1 and vascular cell adhesion molecule are induced by ionizing radiation on lymphatic endothelium(Elsevier, 2016) Etxeberria, I. (Iñaki); Rouzaut, A. (Ana); Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Aristu-Mendioroz, J.J. (José Javier); Barbes-Fernandez, B. (Benigno); Garasa, S. (Saray); Yanguas, A. (Alba); Halin, C. (Cornelia); Melero, I. (Ignacio); Solórzano-Rendón, J.L. (José Luis); Teijeira, A. (Álvaro)Purpose/Objectives The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Materials/Methods Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGFβ in this process anti-TGFβ blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGFβ and /or anti-ICAM1 blocking mAb. Results We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGFβ. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Conclusion Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy.
- New emerging targets in cancer immunotherapy: CD137/4-1BB costimulatory axis(2019) Etxeberria, I. (Iñaki); González-Vaz, J. (Javier); Melero, I. (Ignacio); Teijeira, A. (Álvaro)CD137 (4-1BB) is a surface glycoprotein that belongs to the tumour necrosis factor receptor family (TNFRSF9). Its expression is induced on activation on a number of leucocyte types. Interestingly, for cancer immunotherapy, CD137 becomes expressed on primed T and natural killer (NK) cells, which on ligation provides powerful costimulatory signals. Perturbation of CD137 by CD137L or agonist monoclonal antibodies on activated CD8 T cells protects such antigen-specific cytotoxic T lymphocytes from apoptosis, enhances effector functionalities and favours persistence and memory differentiation. As a consequence, agonist antibodies exert potent antitumour effects in mouse models and the CD137 signalling domain is critical in chimeric antigen receptors (CAR) of CAR T cells approved to be used in the clinic. New formats of CD137 agonist moieties are being clinically developed, seeking potent costimulation targeted to the tumour microenvironment to avoid liver inflammation side effects, that have thus far limited and delayed clinical development.
- mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy(2023) Egea, J. (Josune); Berraondo, P. (Pedro); Cirella, A. (Assunta); Etxeberria, I. (Iñaki); Álvarez-Rodríguez, M. (Maite); González-Gomariz, J. (José); Bolaños, E. (Elixabet); Eguren-Santamaría, I. (Iñaki); Garasa, S. (Saray); Rabinovich, G.A. (Gabriel A.); Hervas-Stubbs, S. (Sandra); González-Vaz, J. (Javier); Melero, I. (Ignacio); Guedan, S. (Sonia); Mariño, K. (Karina); Olivera, I. (Irene); Fernandez-Sanmamed, M. (Miguel); Luri-Rey, C. (Carlos); Teijeira, A. (Álvaro)Interleukin-12 (IL-12) gene transfer enhances the therapeutic potency of adoptive T cell therapies. We previ-ously reported that transient engineering of tumor-specific CD8 T cells with IL-12 mRNA enhanced their sys-temic therapeutic efficacy when delivered intratumorally. Here, we mix T cells engineered with mRNAs to ex-press either single-chain IL-12 (scIL-12) or an IL-18 decoy-resistant variant (DRIL18) that is not functionally hampered by IL-18 binding protein (IL-18BP). These mRNA-engineered T cell mixtures are repeatedly in-jected into mouse tumors. Pmel-1 T cell receptor (TCR)-transgenic T cells electroporated with scIL-12 or DRIL18 mRNAs exert powerful therapeutic effects in local and distant melanoma lesions. These effects are associated with T cell metabolic fitness, enhanced miR-155 control on immunosuppressive target genes, enhanced expression of various cytokines, and changes in the glycosylation profile of surface proteins, enabling adhesiveness to E-selectin. Efficacy of this intratumoral immunotherapeutic strategy is recapitu-lated in cultures of tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells on IL -12 and DRIL18 mRNA electroporation.
- Repurposing infectious disease vaccines for intratumoral immunotherapy(2020) Valsesia-Wittmann, S. (Sandrine); Etxeberria, I. (Iñaki); Gato-Cañas, M. (María); Aznar, M.A. (María Ángela); Marabelle, A. (Aurelien); Melero, I. (Ignacio); Caux, C. (Christophe); Shekarian, T. (Tala); Teijeira, A. (Álvaro)Intratumoral delivery of viruses and virus-associated molecular patterns can achieve antitumor effects that are largely mediated by the elicitation or potentiation of immune responses against the malignancy. Attenuated vaccines are approved and marketed as good manufactiring practice (GMP)-manufactured agents whose administration might be able to induce such effects. Recent reports in mouse transplantable tumor models indicate that the rotavirus, influenza and yellow fever vaccines can be especially suitable to elicit powerful antitumor immunity against cancer following intratumoral administration. These results highlight that intratumoral anti-infectious vaccines can turn cold tumors into hot, and underscore the key role played by virus-induced type I interferon pathways to overcome resistance to immune checkpoint-targeted antibodies.