Minute, L. (Luna)
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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.
- 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.
- 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.
- 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.
- Daratumumab in combination with urelumab to potentiate anti-myeloma activity in lymphocytedeficient mice reconstituted with human NK cells(Informa UK Limited, 2019) Berraondo, P. (Pedro); Zabaleta, A. (Aintzane); Fernández-Sendín, M. (Myriam); López-Botet, M. (Miguel); Perez-Ruiz, E. (Elisabeth); Rodriguez, I. (Inmaculada); Muntasell, A. (Aura); Oñate, C. (Carmen); Lopez, A. (Ascensión); Ochoa, M.C. (María Carmen); Alignani, D. (Diego); Melero, I. (Ignacio); Paiva, B. (Bruno); Perez, G. (Guiomar); Minute, L. (Luna); Fernandez-Sanmamed, M. (Miguel)Daratumumab is an anti-CD38 fully human IgG1 mAb approved for multiple myeloma treatment. One of the proposed mechanisms of action is the induction of antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. NK cells acquire surface CD137 expression in the presence of solid-phase-attached daratumumab and when encountering a daratumumab-coated CD38+ tumor cell line. In this setting, addition of the agonist anti-CD137 mAb urelumab enhances NK-cell activation increasing CD25 expression and IFNɣ production. However, in vitro ADCC is not increased by the addition of urelumab both in 4h or 24h lasting experiments. To study urelumab-increased daratumumab-mediated ADCC activity in vivo, we set up a mouse model based on the intravenous administration of a luciferase-transfected multiple myeloma cell line of human origin, human NK cells and daratumumab to immuno-deficient NSG mice. In this model, intravenous administration of urelumab 24h after daratumumab delayed tumor growth and prolonged mice survival.
- 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.