Álvarez, M. (Maite)
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- Intratumoral neoadjuvant immunotherapy based on the BO-112 viral RNA mimetic(2023) Glez-Vaz, J. (Javier); Berraondo, P. (Pedro); Cirella, A. (Assunta); Azpilikueta, A. (Arantza); González-Gomariz, J. (José); Bolaños, E. (Elixabet); Rodriguez-Ruiz, M.E. (María Esperanza); Garasa, S. (Saray); Gomis, G. (Gabriel); Quintero, M. (Marisol); Molina, C. (Carmen); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Álvarez, M. (Maite); Olivera, I. (Irene); Luri-Rey, C. (Carlos); Teijeira, A. (Álvaro)BO-112 is a poly I:C-based viral mimetic that exerts anti-tumor efficacy when intratumorally delivered in mouse models. Intratumoral BO-112 synergizes in mice with systemic anti-PD-1 mAbs and this combination has attained efficacy in PD1-refractory melanoma patients. We sought to evaluate the anti-tumor efficacy of BO-112 pre-surgically applied in neoadjuvant settings to mouse models. We have observed that repeated intratumoral injections of BO-112 prior to surgical excision of the primary tumor significantly reduced tumor metastasis from orthotopically implanted 4T1-derived tumors and subcutaneous MC38-derived tumors in mice. Such effects were enhanced when combined with systemic anti-PD-1 mAb. The anti-tumor efficacy of this neoadjuvant immunotherapy approach depended on the presence of antigen-specific effector CD8 T cells and cDC1 antigen-presenting cells. Since BO-112 has been successful in phase-two clinical trials for metastatic melanoma, these results provide a strong rationale for translating this pre-surgical strategy into clinical settings, especially in combination with standard-of-care checkpoint inhibitors.
- Impact of prophylactic TNF blockade in the dual PD-1 and CTLA-4 immunotherapy efficacy and toxicity(Shared Science Publishers OG, 2019) Berraondo, P. (Pedro); Perez-Ruiz, E. (Elisabeth); Otano, I. (Itziar); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Álvarez, M. (Maite); Minute, L. (Luna)The TNF blockade therapy is currently a well-established treatment option for a variety of autoimmune diseases such as rheumatoid arthritis (RA), psoriasis or Crohn's disease, given the proinflammatory role of TNF in the course of these diseases. Importantly, TNF neutralization is also used for the treatment of corticosteroid-refractory immune-related adverse events (irAEs) induced by the combined anti-PD-1 and anti-CTLA-4 immunotherapy. The manifestation of these toxicities is an important limiting factor for the successful implementation of the inhibitory checkpoint blockade therapy (ICB), restraining its anti-tumor efficacy. In our recent study (Perez-Ruiz et al., Nature 569(7756): 428-432.), we analyzed the potential impact of prophylactic TNF neutralization therapy in the anti-PD1/CTLA-4 efficacy. Through several mouse models, we demonstrated that TNF neutralization ameliorated ICB-exacerbated colitis in addition to improving ICB-dependent anti-tumor efficacy. Similar results were obtained after prophylactic TNF blockade in graft vs host xenografted mouse models with human immune cells, which showed a reduction in colitis and hepatitis. Importantly, there was a preservation of the immunotherapeutic control of xenografted tumors after ICB treatment. Moreover, TNF and TNF-dependent gene expression is upregulated in the colon mucosa from patients affected by colitis as a side effect of ipilimumab and nivolumab. Our results, thus, provide evidence of the successful combination of prophylactic TNF neutralization with ICB therapy strategy to ameliorate toxicities, while keeping or even ameliorating anti-tumor efficacy. The prophylactic TNF blockade strategy is clinically feasible since excellent TNF inhibitors have been approved for the treatment of autoimmunity and are used for the immune-related serious adverse events in immunotherapy.
- 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.
- IL-2 and Anti-TGF-β Promote NK Cell Reconstitution and Anti-tumor Effects after Syngeneic Hematopoietic Stem Cell Transplantation(2020) Khuat, L.T. (Lam T.); Barao, I. (Isabel); Dunai, C. (Cordelia); Murphy, W.J. (William J.); Álvarez, M. (Maite); Aguilar, E.G. (Ethan G.)The failure of autologous hematopoietic stem cell transplantation (HSCT) has been associated with a profound immunodeficiency that follows shortly after treatment, which renders patients susceptible to opportunistic infections and/or cancer relapse. Thus, given the additional immunosuppressive pathways involved in immune evasion in cancer, strategies that induce a faster reconstitution of key immune effector cells are needed. Natural killer (NK) cells mediate potent anti-tumor effector functions and are the first immune cells to repopulate after HSCT. TGF-β is a potent immunosuppressive cytokine that can impede both the development and function of immune cells. Here, we evaluated the use of an immunotherapeutic regimen that combines low dose of IL-2, an NK cell stimulatory signal, with TGF-β neutralization, in order to accelerate NK cell reconstitution following congenic HSCT in mice by providing stimulatory signals yet also abrogating inhibitory ones. This therapy led to a marked expansion of NK cells and accelerated NK cell maturation. Following HSCT, mature NK cells from the treated recipients displayed an activated phenotype and enhanced anti-tumor responses both in vitro and in vivo. No overt toxicities or adverse effects were observed in the treated recipients. However, these stimulatory effects on NK cell recovery were predicated upon continuous treatment as cessation of treatment led to return to baseline levels and to no improvement of overall immune recovery when assessed at later time-points, indicating strict regulatory control of the NK cell compartment. Overall, this study still demonstrates that therapies that combine positive and negative signals can be plausible strategies to accelerate NK cell reconstitution following HSCT and augment anti-tumor efficacy.
- Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy(Springer Science and Business Media LLC, 2019) Berraondo, P. (Pedro); Llacer, C. (Casilda); Perez-Gracia, J.L. (Jose Luis); Perez-Ruiz, E. (Elisabeth); Rodriguez-Ruiz, M.E. (María Esperanza); Luque, V. (Vanesa) de; Marquez-Rodas, I. (Iván); Otano, I. (Itziar); Molina, C. (Carmeen); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Andrea, C.E. (Carlos Eduardo) de; Belsue, V. (Virginia); Álvarez, M. (Maite); Álvarez, M. (Martina); Minute, L. (Luna); Teijeira, A. (Álvaro)Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.
- Intratumoral co-injection of the poly I:C-derivative BO-112 and a STING agonist synergize to achieve local and distant anti-tumor efficacy(2021) Glez-Vaz, J. (Javier); Berraondo, P. (Pedro); Fan, X. (X.); Fernández-Sendín, M. (Myriam); de Andrea, C. E. (Carlos E.); González-Gomariz, J. (José); Rodriguez-Ruiz, M.E. (María Esperanza); Villalba, M. (María); Quintero, M. (Marisol); Aranda, F. (Fernando); Molina, C. (Carmeen); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Álvarez, M. (Maite); Shen, W. H. (Wen H.); Fernandez-Sanmamed, M. (Miguel); Teijeira, A. (Álvaro)Background BO-112 is a nanoplexed form of polyinosinic:polycytidylic acid that acting on toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5) and protein kinase RNA-activated (PKR) elicits rejection of directly injected transplanted tumors, but has only modest efficacy against distant untreated tumors. Its clinical activity has also been documented in early phase clinical trials. The 5,6-dimethylxanthenone-4-acetic acid (DMXAA) stimulator of interferon genes (STING) agonist shows a comparable pattern of efficacy when used via intratumoral injections. Methods Mice subcutaneously engrafted with bilateral MC38 and B16.OVA-derived tumors were treated with proinflammatory immunotherapy agents known to be active when intratumorally delivered. The combination of BO-112 and DMXAA was chosen given its excellent efficacy and the requirements for antitumor effects were studied on selective depletion of immune cell types and in gene-modified mouse strains lacking basic leucine zipper ATF-like transcription factor 3 (BATF3), interferon-α/β receptor (IFNAR) or STING. Spatial requirements for the injections were studied in mice bearing three tumor lesions. Results BO-112 and DMXAA when co-injected in one of the lesions of mice bearing concomitant bilateral tumors frequently achieved complete local and distant antitumor efficacy. Synergistic effects were contingent on CD8 T cell lymphocytes and dependent on conventional type 1 dendritic cells, responsiveness to type I interferon (IFN) and STING function in the tumor-bearing host. Efficacy was preserved even if BO-112 and DMXAA were injected in separate lesions in a manner able to control another untreated third-party tumor. Efficacy could be further enhanced on concurrent PD-1 blockade. Conclusion Clinically feasible co-injections of BO-112 and a STING agonist attain synergistic efficacy able to eradicate distant untreated tumor lesions.
- Cellular cytotoxicity is a form of immunogenic cell death(2020) Berraondo, P. (Pedro); Etxeberria, I. (Iñaki); Azpilikueta, A. (Arantza); Perez-Gracia, J.L. (Jose Luis); Sanchez-Paulete, A.R. (Alfonso R.); Bolaños, E. (Elixabet); Rodriguez-Ruiz, M.E. (María Esperanza); Garasa, S. (Saray); Otano, I. (Itziar); Casares, N. (Noelia); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Álvarez, M. (Maite); Minute, L. (Luna); Teijeira, A. (Álvaro)Background The immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or natural killer (NK) lymphocytes, is actually immunogenic currently remains unknown. Methods In this study, tumor cells were killed by antigenspecific T-cell receptor (TCR) transgenic CD8 T cells or activated NK cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumorassociated antigen and by class I major histocompatibility complex (MHC)-multimer stainings. Mice deficient in Batf3, Ifnar1 and Sting1 were used to study mechanistic requirements. Results We observe in cocultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. Ovalbumin (OVA)-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100-specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+ EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient in Batf3- dependent dendritic cells (conventional dendritic cells type 1, cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+ T lymphocytes. Conclusion These results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death.
- Indirect Impact of PD-1/PD-L1 Blockade on a Murine Model of NK Cell Exhaustion(2020) Berraondo, P. (Pedro); Negrin, R.S. (Robert S.); Simonetta, F. (Federico); Morrison, A.R. (Alyssa R.); Pierini, A. (Antonio); Baker, J. (Jeanette); Álvarez, M. (Maite); Wenokur, A.S. (Arielle S.)The induction of exhaustion on effector immune cells is an important limiting factor for cancer immunotherapy efficacy as these cells undergo a hierarchical loss of proliferation and cytolytic activity due to chronic stimulation. Targeting PD-1 has shown unprecedented clinical benefits for many cancers, which have been attributed to the prevention of immune suppression and exhaustion with enhanced anti-tumor responses. In this study, we sought to evaluate the role of the PD-1/PD-L1 pathway in murine natural killer (NK) cell activation, function, and exhaustion. In an in vivo IL-2-dependent exhaustion mouse model, neutralization of the PD-1/PD-L1 pathway improved NK cell activation after chronic stimulation when compared to control-treated mice. These cells displayed higher proliferative capabilities and enhanced granzyme B production. However, the blockade of these molecules during long-term in vitro IL-2 stimulation did not alter the progression of NK cell exhaustion (NCE), suggesting an indirect involvement of PD-1/PD-L1 on NCE. Given the expansion of CD8 T cells and regulatory T cells (Tregs) observed upon acute and chronic stimulation with IL-2, either of these two populations could influence NK cell homeostasis after PD-L1/PD-1 therapy. Importantly, CD8 T cell activation and functional phenotype were indeed enhanced by PD-1/PD-L1 therapy, particularly with anti-PD-1 treatment that resulted in the highest upregulation of CD25 during chronic stimulation and granted an advantage for IL-2 over NK cells. These results indicate a competition for resources between NK and CD8 T cells that arguably delays the onset of NCE rather than improving its activation during chronic stimulation. Supporting this notion, the depletion of CD8 T cells reversed the benefits of PD-1 therapy on chronically stimulated NK cells. These data suggest a bystander effect of anti-PD1 on NK cells, resulting from the global competition that exists between NK and CD8 T cells for IL-2 as a key regulator of these cells’ activation. Thus, achieving an equilibrium between these immune cells might be important to accomplish long-term efficacy during anti-PD-1/IL-2 therapy.
- Intratumoral BO-112 in combination with radiotherapy synergizes to achieve CD8 T-cell-mediated local tumor control(2023) Escuin-Ordinas, H. (Helena); Perez-Gracia, J.L. (Jose Luis); Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Arbea-Moreno, L. (Leire); Garate-Soraluze, E. (Eneko); Serrano-Mendioroz, I. (Irantzu); Quintero, M. (Marisol); Melero, I. (Ignacio); Diaz-Pascual, V. (Victor); Barrio-Alonso, C. (Celia); Álvarez, M. (Maite); Sánchez-Mateos, P. (Paloma); Fernandez-Sanmamed, M. (Miguel)BackgroundRadioimmunotherapy combines irradiation of tumor lesions with immunotherapy to achieve local and abscopal control of cancer. Most immunotherapy agents are given systemically, but strategies for delivering immunotherapy locally are under clinical scrutiny to maximize efficacy and avoid toxicity. Local immunotherapy, by injecting various pathogen-associated molecular patterns, has shown efficacy both preclinically and clinically. BO-112 is a viral mimetic based on nanoplexed double-stranded RNA (poly I:C) which exerts immune-mediated antitumor effects in mice and humans on intratumoral delivery. BO-112 and focal irradiation were used to make the proof-of-concept for local immunotherapy plus radiation therapy combinations.MethodsMurine transplantable tumor cell lines (TS/A, MC38 and B16-OVA) were used to show increased immunogenic features under irradiation, as well as in bilateral tumor models in which only one of the lesions was irradiated or/and injected with BO-112. Flow cytometry and multiplex tissue immunofluorescence were used to determine the effects on antitumor immunity. Depletions of immune cell populations and knockout mice for the IFNAR and BATF-3 genes were used to delineate the immune system requirements for efficacy.ResultsIn cultures of TS/A breast cancer cells, the combination of irradiation and BO-112 showed more prominent features of immunogenic tumor cell death in terms of calreticulin exposure. Injection of BO-112 into the tumor lesion receiving radiation achieved excellent control of the treated tumor and modest delays in contralateral tumor progression. Local effects were associated with more prominent infiltrates of antitumor cytotoxic tumor lymphocytes (CTLs). Importantly, local irradiation plus BO-112 in one of the tumor lesions that enhanced the therapeutic effects of radiotherapy on distant irradiated lesions that were not injected with BO-112. Hence, this beneficial effect of local irradiation plus BO-112 on a tumor lesion enhanced the therapeutic response to radiotherapy on distant non-injected lesions.ConclusionThis study demonstrates that local BO-112 immunotherapy and focal irradiation may act in synergy to achieve local tumor control. Irradiation plus BO-112 in one of the tumor lesions enhanced the therapeutic effects on distant irradiated lesions that were not injected with BO-112, suggesting strategies to treat oligometastatic patients with lesions susceptible to radiotherapy and with at least one tumor accessible for repeated BO-112 intratumoral injections.