Lopez-Diaz-de-Cerio, A. (Ascensión)
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- Induction of multiepitopic and long-lasting immune responses against tumour antigens by immunization with peptides, DNA and recombinant adenoviruses expressing minigenes(Blackwell, 2009) Borras-Cuesta, F. (Francisco); Lopez-Diaz-de-Cerio, A. (Ascensión); Durantez, M. (Maika); Casares, N. (Noelia); Prieto, J. (Jesús); Huarte, E. (Eduardo); Lopez-Vazquez, A.B. (Ana B.); Sarobe, P. (Pablo); Lasarte, J.J. (Juan José)The development of immunization strategies to induce strong and multiepitopic T-cell responses against tumour antigens is needed for anti-tumour immunotherapy. However, a common finding after immunization with complex antigens is the preferential induction of immune responses against immunodominant epitopes. In this study, with the aim of inducing multiepitopic responses against several common tumour antigens, we have designed a minigene construct encoding four human leucocyte antigen (HLA)-A2-restricted epitopes belonging to tumour antigens CEA (CEA-691 and CEA-571), MAGE2 (MAGE2-157) and MAGE3 (MAGE3-112), as well as the universal PADRE epitope recognized by T helper lymphocytes. To optimize the activation of immune responses against these epitopes, we have used different antigen formats (short peptides encompassing individual epitopes and DNA plasmids or adenoviral constructs expressing the minigene) in single or combined immunization schedules. A single immunization with either DNA plasmid or recombinant adenovirus induced a monospecific immune response against the immunodominant epitope CEA-571, whereas immunization with the peptide pool induced responses against all epitopes. Combination of peptide priming followed by a boost with the plasmid and the recombinant adenovirus expressing the minigene induced stronger, multi-specific and long-lasting immune responses, overcoming the immunodominance imposed by the main T-cell epitope. Moreover, these combined immunization strategies were able to induce responses that were able to recognize Mel624 HLA-A2+ tumour cells expressing MAGE2. These results suggest that heterologous immunization strategies combining peptides and DNA or recombinant adenoviruses can be useful to broaden the specificity and enhance the efficacy of subunit vaccines.
- CD4+/CD25+ regulatory cells inhibit activation of tumor-primed CD4+ T cells with IFN-gamma-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination(American Association of Immunologists, 2003) Dotor, J. (Javier); Lopez-Diaz-de-Cerio, A. (Ascensión); Casares, N. (Noelia); Melero, I. (Ignacio); Sarobe, P. (Pablo); Arribillaga, L. (Laura); Lasarte, J.J. (Juan José)CD25(+) regulatory T (T reg) cells suppress the activation/proliferation of other CD4(+) or CD8(+) T cells in vitro. Also, down-regulation of CD25(+) T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25(+) T reg cells allows the host to induce both CD4(+) and CD8(+) antitumoral responses following tumor challenge. Simultaneous depletion of CD25(+) and CD8(+) cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4(+) T cells, which emerged in the absence of CD25(+) T reg cells, were able to reject CT26 colon cancer cells, a MHC class II-negative tumor. The antitumoral effect mediated by CD4(+) T cells was dependent on IFN-gamma production, which exerted a potent antiangiogenic activity. The capacity of the host to mount this antitumor response is lost once the number of CD25(+) T reg cells is restored over time. However, CD25(+) T reg cell depletion before immunization with AH1 (a cytotoxic T cell determinant from CT26 tumor cells) permits the induction of a long-lasting antitumoral immune response, not observed if immunization is conducted in the presence of regulatory cells. A study of the effect of different levels of depletion of CD25(+) T reg cells before immunization with the peptide AH1 alone, or in combination with a Th determinant, unraveled that Th cells play an important role in overcoming the suppressive effect of CD25(+) T reg on the induction of long-lasting cellular immune responses.
- Engineering Th determinants for efficient priming of humoral and cytotoxic T cell responses(Oxford University Press, 2003) Borras-Cuesta, F. (Francisco); Lopez-Diaz-de-Cerio, A. (Ascensión); Ruiz, M. (Marta); Casares, N. (Noelia); Prieto, J. (Jesús); Sarobe, P. (Pablo); Lasarte, J.J. (Juan José)To engineer T(h) determinants (THd) to prime help for humoral or cytotoxic T cell responses, we modified ovalbumin [OVA(323-337)] and myoglobin [MYO(106-118)] eliciting T(h)1 and T(h)0 cytokine profiles respectively. Residues along the sequence of both THd were replaced with amino acids representative of different families. Replacements at positions P-1 and P5 pointing to the TCR in both THd afforded higher levels of IFN-gamma and IL-4 production. Peptides eliciting different proportions of IFN-gamma and IL-4 were co-immunized with a peptide hapten or a T cytotoxic determinant (TCd) respectively. OVA(323-337)- and MYO(106-118)-derived peptides afforded the best THd for the induction of cytotoxic T lymphocyte (CTL) and anti-hapten antibodies respectively. IFN-gamma and IL-4, primed by MYO(106-118)-derived peptides, correlated significantly with antibody production against the hapten (P < 0.05 for IFN-gamma and P < 0.05 for IL-4). Interestingly, two peptides derived from OVA(323-337), 323G and 327G, which induced the clearest T(h)2 cytokine profiles, were not the most efficient to prime cell help for the induction of anti-hapten antibodies. For CTL induction, OVA(323-337)-derived peptides, inducing a T(h)1-like profile, required a lower dose (5 nmol) than T(h)0 peptides (50 nmol). The dose of 50 nmol was detrimental for T(h)1-like peptides. Interestingly, IFN-gamma primed by the THd correlated significantly with that induced by the TCd (P < 0.01).
- Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins(American Society for Microbiology, 2002) Labarga, P. (Pablo); Baixeras, E. (Elena); Borras-Cuesta, F. (Francisco); Lopez-Diaz-de-Cerio, A. (Ascensión); Casares, N. (Noelia); Prieto, J. (Jesús); Sarobe, P. (Pablo); Garcia, N. (Nicolás); Lasarte, J.J. (Juan José)Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4(+) T cells responding to HCV core in patients with chronic HCV infection. However, CD4(+) response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4(+) response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.
- Final results regarding the addition of dendritic cell vaccines to neoadjuvant chemotherapy in early HER2-negative breast cancer patients: clinical and translational analysis(Sage Journals, 2021) Hato-Álvaro, L. (Laura); Rodríguez-Espiteri, N. (Natalia); Pérez-Solans, B. (Belén); Sanchez-Bayona, R. (Rodrigo); Lopez-Diaz-de-Cerio, A. (Ascensión); Urrizola-Martínez, A. (Amaia); Toledo, E. (Estefanía); Santisteban, M. (Marta); Inoges, S. (Susana); Salgado, E. (Esteban); Mejías-Sosa, L.D. (Luis D.); Idoate, M.A. (Miguel Ángel); Olartecoechea, B. (Begoña)Background:Primary breast cancer (BC) has shown a higher immune infiltration than the metastatic disease, justifying the optimal scenario for immunotherapy. Recently, neoadjuvant chemotherapy (NAC) combined with immune checkpoint inhibitors has demonstrated a gain in pathological complete responses (tpCR) in patients with BC. The aim of our study is to evaluate the safety, feasibility, and efficacy of the addition of dendritic cell vaccines (DCV) to NAC in HER2-negative BC patients. Methods:Thirty-nine patients with early BC received DCV together with NAC conforming the vaccinated group (VG) and compared with 44 patients as the control group (CG). All patients received anthracyclines and taxanes-based NAC (ddECx4→Dx4) followed by surgery ± radiotherapy ± hormonotherapy. Results:The tpCR rate was 28.9% in the VG and 9.09% in the CG (p = 0.03). Pathological CR in the triple negative (TN) BC were 50.0% versus 30.7% (p = 0.25), 16.6% versus 0% in luminal B (p = 0.15), and none among luminal A patients in VG versus CG, respectively. Impact of DCV was significantly higher in the programmed cell death ligand 1 (PD-L1) negative population (p < 0.001). PD-L1 expression was increased in patients with residual disease in the VG as compared with the CG (p < 0.01). No grade ⩾3 vaccine-related adverse events occurred. With a median follow-up of 8 years, no changes were seen in event-free survival or overall survival. Phenotypic changes post DCV in peripheral blood were observed in myeloid-derived suppressor cells (MDSC), NK, and T cells. Increase in blood cell proliferation and interferon (IFN)-γ production was detected in 69% and 74% in the VG, respectively. Humoral response was also found. Clonality changes in TCR-β repertoire were detected in 67% of the patients with a drop in diversity index after treatment. Conclusion:The combination of DCV plus NAC is safe and increases tpCR, with a significant benefit among PD-L1-negative tumors. DCV modify tumor milieu and perform cellular and humoral responses in peripheral blood with no impact in outcome. Trial registration:ClinicalTrials.gov number: NCT01431196. EudraCT 2009-017402-36.
- Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma(Oxford University Press, 2010) Klimyuk, V. (V.); Lopez-Diaz-de-Cerio, A. (Ascensión); Lenz, J. (J.); Fröde, R. (R.); Thieme, F. (F.); Tuse, D. (D.); Inoges, S. (Susana); Villanueva, H. (Helena); McCormick, A. (A.); Kandzia, R. (R.); Herz, S. (S.); Bendandi, M. (Maurizio); Butler-Ransohoff, J.E. (J.E.); Marillonnet, S. (S.); Vancanneyt, G. (G.); Soria, E. (Elena); Nickstadt, A. (A.); Gleba, Y. (Y.)BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.
- Imiquimod enhances the systemic immunity attained by local cryosurgery destruction of melanoma lesions.(Nature Publishing Group, 2007) Lopez-Diaz-de-Cerio, A. (Ascensión); Inoges, S. (Susana); Olmo-López, J. (Julio) del; Melero, I. (Ignacio); Bendandi, M. (Maurizio); Redondo-Bellón, P. (Pedro); Marquina, M. (Miren)Melanoma lesions can be frozen in vivo, resulting in necrotic death of malignant cells and in tumor antigen release suitable for cross-presentation by professional antigen-presenting cells. Imiquimod is a small molecule with adjuvant pro-inflammatory effects that can be topically delivered as a cream. Local cryosurgery of B16/ovalbumin (OVA)-derived subcutaneous tumor nodules leads to curative destruction of the lesions. If imiquimod is repeatedly applied on the cryo-treated lesion, a conspicuous, leukocyte-rich inflammatory infiltrate appears during the days following treatment. Mice treated by cryosurgery plus imiquimod rejected rechallenges of B16/OVA in 90% of the cases, whereas cryosurgery alone failed to prevent tumor grafting in 70% of the cases. The combination treatment of B16/OVA tumors was also able to protect 60% of the mice against outgrowth of a lethal dose of non-transfected B16 tumor cells. Addition of imiquimod to cryosurgery results in increases of the cellular immune response against tumor antigens as measured by in vitro IFN-gamma production and T-cell proliferation in response to OVA. The potent memory response is not only directed against the OVA epitope, but also toward a broader range of B16 antigens. Our data indicate that these combined treatments turn the treated tumor lesion into an autologous tumor vaccine, which is even able to cause vitiligo in several cases. These preclinical data and the simplicity of the procedures warrant the design of a pilot clinical trial.
- Vaccination with an adenoviral vector encoding hepatitis C virus (HCV) NS3 protein protects against infection with HCV-recombinant vaccinia virus(Elsevier, 2002) Paranhos-Baccala, G. (Glaucia); Bruña-Romero, O. (Oscar); Vales, A. (África); Borras-Cuesta, F. (Francisco); Lopez-Diaz-de-Cerio, A. (Ascensión); Casares, N. (Noelia); Prieto, J. (Jesús); Sarobe, P. (Pablo); Gorraiz, M. (Marta); Arribillaga, L. (Laura); Ruiz, J. (Juan); Lasarte, J.J. (Juan José)Cellular immune response plays an important role in the clearance of hepatitis C virus (HCV). Thus, development of efficient ways to induce anti-viral cellular immune responses is an important step toward prevention and/or treatment of HCV infection. With this aim, we have constructed a replication-deficient recombinant adenovirus expressing HCV NS3 protein (RAdNS3). The efficacy of RAdNS3 was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV-polyprotein (vHCV1-3011). Immunisation with 10(9)pfu of RAdNS3 induced anti-NS3 humoral, T helper and T cytotoxic responses. We identified eight epitopes recognised by IFN-gamma producing cells, five of them exhibiting lytic activity. Moreover, we show that RAdNS3 immunised mice were protected against challenge with vHCV1-3011 and that this protection was mediated by CD8(+) cells. In conclusion, our results suggest that adenoviral vectors encoding NS3 might be useful for the induction of prophylactic and/or therapeutic anti-HCV immunity.
- Variations in “rescuability” of immunoglobulin molecules from different forms of human lymphoma: implications for anti-idiotype vaccine development(Elsevier, 2004) Lopez-Diaz-de-Cerio, A. (Ascensión); Inoges, S. (Susana); Villanueva, H. (Helena); Bendandi, M. (Maurizio); Zabalegui, N. (Natalia); Rodriguez-Calvillo, M. (Mercedes)Idiotypic (Id) vaccination has shown promising results in patients with follicular lymphoma (FL). However, it still remains unclear whether the same approach might be suitable for the treatment of other B-cell malignancies. For this reason, we recently performed an interim analysis of patients proposed to receive this treatment at our center. The feasibility of employing idiotype vaccines was evaluated for five different B-cell malignancies in their first relapse, both in terms of induction and fusion, as well as overall treatment. Our data suggest that, unlike follicular lymphoma (87%), this approach is not feasible to treat other B-cell malignancies (0–20%) such as mantle cell, small lymphocytic, diffuse large cell and Burkitt’s lymphoma (P < 0.01). The main difficulties encountered were technical problems related to the survival of idiotype-producing hybridomas (83%) and the early loss of idiotype production by growing hybridomas (17%). However, it remains possible that an idiotype vaccine might still be produced through molecular means for most, if not all cases of relapsing B-cell malignancies.
- Anti-idiotype antibodies in cancer treatment(Nature Publishing Group, 2007) Lopez-Diaz-de-Cerio, A. (Ascensión); Inoges, S. (Susana); Bendandi, M. (Maurizio); Zabalegui, N. (Natalia); Rodriguez-Calvillo, M. (Mercedes)As a cancer immunotherapy tool, idiotypes (Ids) have been used in different ways over the last three decades, depending on the actual human tumor cell target. It all started with passive, monoclonal, anti-Id antibody treatment of B-cell lymphoma, a setting in which results were tantalizing, but logistics unsustainable. It then moved toward the development of anti-Id vaccines for the treatment of the same tumors, a setting in which we have recently provided the first formal proof of principle of clinical benefit associated with the use of a human cancer vaccine. Meanwhile, it also expanded in the direction of exploiting the antigenic mimicry of some Ids with Id-unrelated, tumor-associated antigens for the immunotherapy of a number of solid tumors, a setting in which clinical results are still far from being consolidated. All in all, over the years Id-based immunotherapy has paved the way for a number of seminal therapeutic improvements for cancer patients, including the development of most if not all Id-unrelated monoclonal antibodies that have recently revolutionized the field.