Rodriguez-Calvillo, M. (Mercedes)

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    Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells
    (Elsevier, 2001) Mazzolini, G. (Guillermo); Duarte, M. (Marina); Gabari, I. (Izaskun); Rifon, J. J. (Jose J.); Rocha, E. (Eduardo); Melero, I. (Ignacio); Prieto, J. (Jesús); Rodriguez-Calvillo, M. (Mercedes)
    OBJECTIVE: With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. MATERIALS AND METHODS: One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. RESULTS: EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. CONCLUSIONS: Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura.
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    Acquired potential N-glycosylation sites within the tumor-specific immunoglobulin heavy chains of B-cell malignancies
    (Pensiero Scientifico / Ferrata Storti Foundation, 2004) Martin-Algarra, S. (Salvador); Lopez-Diaz-de-Cerio, A. (Ascensión); Rocha, E. (Eduardo); Inoges, S. (Susana); Perez-Calvo, J. (Javier); Hernandez, M. (Milagros); Garcia-Foncillas, J. (Jesús); Bendandi, M. (Maurizio); Zabalegui, N. (Natalia); Rodriguez-Calvillo, M. (Mercedes)
    Background and Objectives. Among B-cell malignancies, follicular lymphomas (FL) more frequently show acquired, potential N-glycosylation sites (AGS) within tumor-specific immunoglobulin. The aim of this study was to extend this observation and to evaluate the pattern of presentation of AGS within five different forms of B-cell lymphoma. Design and Methods. We sequenced the tumor-specific immunoglobulin heavy chain variable region fragment, including complementarity-determining regions 2 and 3, of forty-seven consecutive patients with a B-cell malignancy enrolled in idiotype vaccine clinical trials. This sequencing approach is known to allow the identification of most AGS. We then statistically analyzed differences in presentation pattern, in terms of tumor histology, immunoglobulin isotype, AGS location and amino acid composition. Results. All twenty-four FL cases presented with at least one AGS, whereas the vast majority of four B-cell lymphoma types other than FL did not. The non- FL group of tumors included four cases of Burkitt’s lymphoma, six of diffuse large cell lymphoma, seven mantle cell lymphomas and six small lymphocytic lymphomas. Most IgM-bearing follicular lymphoma cases featured their AGS within complementarity-determining region 2, as opposed to those bearing an IgG, which mostly displayed the AGS within complementarity- determining region 3. The vast majority of AGS located within either complementarity- determining region ended with a serine residue, whereas those located within framework regions mostly featured threonine as the last amino acid residue. Interpretation and Conclusions. In our series, all cases of FL had AGS within their tumor-specific immunoglobulin heavy chain variable regions. In contrast, most B-cell malignancies other than FL did not. Further studies are warranted in order to establish the possible meaning of these findings in terms of disease pathogenesis, their diagnostic value in doubtful cases and their potential implications for immunotherapy.
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    Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12
    (Elsevier, 2002) Berraondo, P. (Pedro); Mazzolini, G. (Guillermo); Duarte, M. (Marina); Qian, C. (Cheng); Melero, I. (Ignacio); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Rodriguez-Calvillo, M. (Mercedes)
    OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12.
  • Prolonged idiotypic vaccination against follicular lymphoma
    (Informa Healthcare, 2009) Pastor, F. (Fernando); Orfao, A. (Alberto); Lopez-Diaz-de-Cerio, A. (Ascensión); Rodriguez-Caballero, A. (Arancha); Panizo, C. (Carlos); Inoges, S. (Susana); Villanueva, H. (Helena); Bendandi, M. (Maurizio); Suarez, L. (Lilia); Soria, E. (Elena); Zabalegui, N. (Natalia); Rodriguez-Calvillo, M. (Mercedes)
    During the last 2 decades, idiotypic vaccination has provided proof of principle of biological efficacy, clinical efficacy and clinical benefit in small follicular lymphoma trials. However, with the exception of anecdotal reports, most patients have received no more than 10 doses of their customised idiotype (Id) vaccine. Therefore, it is not known whether prolonged usage of idiotypic vaccination is safe. Since 2002, 18 previously treated patients with follicular lymphoma have received extended idiotypic vaccination at our institution outside clinical trials. Vaccination was provided as a compassionate alternative to no further treatment, and was meant to be stopped only upon complete consumption of the available patient- and tumor-specific vaccine [Id-keyhole limpet hemocyanin + granulocyte-macrophage colony-stimulating factor (Id-KLH + GM-CSF)], or in case of disease relapse or any serious non-local toxicity. So far, 18 patients have received an average of 18 doses of Id vaccine (median: 17; mean: 18; range: 10-31). Eleven patients are still actively receiving idiotypic vaccination: some of them are now over more than 6 years. Toxicity has been systematically negligible and mostly local. No patient has abandoned the vaccination program because of toxicity. Prolonged idiotypic vaccination with the soluble protein Id-KLH + GM-CSF formulation is safe and well tolerated.
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    Past, present and future of anti-idiotype vaccination
    (Universidad de Navarra. Facultad de Medicina, 2004) Lopez-Diaz-de-Cerio, A. (Ascensión); Rocha, E. (Eduardo); Panizo, C. (Carlos); Inoges, S. (Susana); Perez-Calvo, J. (Javier); Hernandez, M. (Milagros); Melero, I. (Ignacio); Bendandi, M. (Maurizio); Prosper-Cardoso, F. (Felipe); Sanchez-Ibarrola, A. (Alfonso); Zabalegui, N. (Natalia); Rodriguez-Calvillo, M. (Mercedes)
    Cancer vaccines are conceived as therapeutic tools, in contrast to the prophylactic vaccines used to fight against infectious diseases. Among the most potent therapeutic vaccines, anti-idiotype vaccination is directed against the tumor idiotype, the only well-characterized tumor antigen displayed in neoplastic B-cells. Anti-idiotype vaccines have demonstrated clinical benefit against follicular lymphoma and are currently being evaluated in two different phase III clinical trials. Additional emerging strategies, which include the use of dendritic cells and the production of vaccines via molecular means will surely allow us to draw important conclusions concerning the treatment of cancer patients.
  • Combined vaccination with idiotype-pulsed allogeneic dendritic cells and soluble protein idiotype for multiple myeloma patients relapsing after reduced-intensity conditioning allogeneic stem cell transplantation.
    (Informa Healthcare, 2006) Perez-Simon, J.A. (José Antonio); Orfao, A. (Alberto); Lopez-Diaz-de-Cerio, A. (Ascensión); Garcia-Montero, A. (A.); Rodriguez-Caballero, A. (Arancha); Almeida, J. (J.); Inoges, S. (Susana); Giraldo, P. (P.); Bendandi, M. (Maurizio); Zabalegui, N. (Natalia); San-Miguel, J.F. (Jesús F.); Rodriguez-Calvillo, M. (Mercedes)
    BACKGROUND AND OBJECTIVE: To combine the use of idiotype-pulsed allogeneic dendritic cells (alloDC) and soluble protein Id conjugated with KLH (Id-KLH) in a vaccine strategy for multiple myeloma (MM). DESIGN AND METHODS: Four MM patients received the combined vaccine after having experienced disease relapse/progression following reduced intensity conditioning (RIC) allogeneic stem cell transplantation (alloSCT) and failure to rescue therapy with donor lymphocyte infusion or chemotherapy (CHT). RESULTS: Vaccination was well tolerated and induced an anti-KLH antibody response in all 4 patients as well as substantial cell proliferation. In contrast, no case showed similar effects against either tumor-specific Id or irrelevant isotype control immunoglobulins (Ig). In turn, vaccination was associated with modulation of biological responses linked to both inflammatory and T-cell activation, with secretion of effector Th1 cytokines. In particular, an important increase in the spontaneous ex vivo secretion of TNFalpha, IL-6 and IFNgamma as well as IL-2 and IL-10 was frequently observed prior to the fourth vaccination. Moreover, in vitro stimulation with Id-KLH and Id-KLH plus alloDC, but not with alloDC alone was associated with an enhanced number of TNF-alpha+ T-cells and an increased secretion of IFNgamma and IL-2 before the third and fourth vaccination. From a clinical standpoint, 2 patients had a transient response and 1 has stable disease after stopping vaccination, while 3 of them ultimately progressed. INTERPRETATION AND CONCLUSIONS: The results show for the first time that the use of Id-pulsed alloDC following RIC alloSCT is safe and feasible. However, crucial strategy improvements are warranted to possibly achieve clinical benefit.
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    An anti-ICAM-2 (CD102) monoclonal antibody induces immune-mediated regressions of transplanted ICAM-2-negative colon carcinomas
    (American Association for Cancer Research, 2002) Albar, J.P. (Juan P.); Mazzolini, G. (Guillermo); Gabari, I. (Izaskun); Melero, I. (Ignacio); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Camafeita, E. (Emilio); Relloso, M. (Miguel); Schmitz, V. (Volker); Rodriguez-Calvillo, M. (Mercedes); Corbi, A.L. (Angel L.)
    Monoclonal antibodies (mAbs) can mediate antitumor effects by indirect mechanisms involving antiangiogenesis and up-regulation of the cellular immune response rather than by direct tumor cell destruction. From mAbs raised by immunization of rats with transformed murine endothelial cells, a mAb (EOL4G8) was selected for its ability to eradicate a fraction of established colon carcinomas that did not express the EOL4G8-recognized antigen. The antigen was found to be ICAM-2 (CD102). Antitumor effects of EOL4G8, which required a functional T-cell compartment, were abrogated by depletion of CD8(+) cells and correlated with antitumor CTL activity, whereas only a mild inhibition of angiogenesis was observed. Interestingly, we found that EOL4G8 acting on endothelial ICAM-2 markedly enhances leukotactic factor activity-1-independent adhesion of immature dendritic cells to endothelium-an effect that is at least in part mediated by DC-SIGN (CD209).
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    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.
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    Feasibility of idiotype vaccination in relapsed B-cell malignancies
    (Ferrata Storti Foundation, 2003) Lopez-Diaz-de-Cerio, A. (Ascensión); Rocha, E. (Eduardo); Panizo, C. (Carlos); Inoges, S. (Susana); Perez-Calvo, J. (Javier); Hernandez, M. (Milagros); Cuesta, B. (Braulia); Bendandi, M. (Maurizio); Zabalegui, N. (Natalia); Rodriguez-Calvillo, M. (Mercedes)