Combined immunotherapy encompassing intratumoral poly-ICLC, dendritic-cell vaccination and radiotherapy in advanced cancer patients.

Background: Combination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methods: In this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24 h with poly-ICLC (Hiltonol), TNF-α and IFN-α. On days +8 and +10 of each cycle, patients received intratumoral image-guided 0.25 mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600 mg/m2 was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre- and post-treatment PBMC from patients achieving durable stable disease (SD) were studied by IFNγ ELISPOT-assays responding to tumor-lysate loaded DC and by TCRß sequencing. Results: Combined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABR+ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-ß and IFN-α mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1ß concentrations, especially in patients presenting SD. IFNγ-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. Conclusions: This radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy.

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma.

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.

[Idiotype vaccines in the treatment of follicular lymphoma: current status and future perspectives]. / Vacuna idiotípica en el tratamiento del linfoma folicular: situación actual y perspectivas futuras.

Follicular lymphoma is the second most prevalent non-Hodgkin lymphoma, representing 20% of all lymphomas. Follicular lymphoma is an indolent disease with a slow progression in which, although exhibiting a good response to treatment, relapse is very frequent and complete remission is not easy to maintain. Therefore, the disease is regarded as incurable. The search for new therapeutic strategies, together with a better understanding of the immune system, has led to the emergence of a new treatment named immunotherapy. Follicular lymphoma is a malignancy suitable for this kind of treatment given the fact that it is characterized by presenting a unique tumour-specific antigen: the idiotype of the monoclonal immunoglobulin displayed on the membrane of tumour cells. Several studies have been conducted to test immunotherapy as complementary to conventional treatment. In a previous study by our group, a clear benefit was evident is obtained after idiotypic vaccination, when an adequate immunization of the patient is obtained, in comparison to chemotherapy alone. In this sense, analysis is needed of whether idiotypic vaccination can produce not only long-lasting and complete remission, but even cure. It would be of great interest to consider an optimisation of the experimental design of clinical trials, an improvement of vaccine production, and the study of the molecular mechanisms of the tumour cell which modify the target immunoglobulin.

Vacuna idiotípica en el tratamiento del linfoma folicular: situación actual y perspectivas futuras / Idiotype vaccines in the treatment of follicular lymphoma: current status and future perspectives

El linfoma folicular (LF) está considerado como elsegundo tipo de linfoma no-Hodgkin más común, representandomás del 20% del total de los linfomas. Es unaenfermedad de progresión lenta y curso indolente enla que, a pesar de la buena respuesta al tratamiento,las recaídas son muy frecuentes y cada vez es más difícilconseguir respuestas completas. Por ello, se puedeconsiderar que hasta el momento, el LF es incurable.La búsqueda continua de nuevas estrategias terapéuticasen enfermedades neoplásicas, junto con un mejorconocimiento del sistema inmunitario, ha llevado ala aparición de una nueva disciplina, conocida con elnombre de inmunoterapia, que aprovecha la capacidaddel sistema inmunitario de atacar lo extraño sin dañarlo propio. El LF es un tumor muy apropiado para estetipo de tratamiento por presentar un antígeno específicode tumor: el idiotipo de la inmunoglobulina monoclonalexpresada en la membrana de todas las célulastumorales. Se han realizado diversos estudios en losque se ha probado la inmunoterapia como tratamientocomplementario al tratamiento convencional. Recientemente,nuestro grupo ha publicado un estudio en el quese observa claramente que los resultados que se obtienentras la vacunación idiotípica, cuando se consigue lainmunización adecuada del paciente, son mejores quelos obtenidos con quimioterapia sola. En este sentido,es necesario seguir investigando para aclarar si la vacunaciónidiotípica pudiera no sólo mantener remisionescompletas duraderas en los pacientes vacunados, sinoincluso conseguir la curación de los mismos. Por ello,resulta interesante abordar un mejor planteamiento delos ensayos clínicos, la mejora de la producción de lavacuna y el estudio de mecanismos de la célula tumoralcapaces de modificar la inmunoglobulina específica del tumor(AU)

Follicular lymphoma is the second most prevalentnon-Hodgkin lymphoma, representing 20% of all lymphomas.Follicular lymphoma is an indolent diseasewith a slow progression in which, although exhibitinga good response to treatment, relapse is very frequentand complete remission is not easy to maintain. Therefore,the disease is regarded as incurable. The searchfor new therapeutic strategies, together with a betterunderstanding of the immune system, has led to theemergence of a new treatment named immunotherapy.Follicular lymphoma is a malignancy suitable for thiskind of treatment given the fact that it is characterizedby presenting a unique tumour-specific antigen: theidiotype of the monoclonal immunoglobulin displayedon the membrane of tumour cells. Several studies havebeen conducted to test immunotherapy as complementaryto conventional treatment. In a previous study byour group, a clear benefit was evident is obtained afteridiotypic vaccination, when an adequate immunizationof the patient is obtained, in comparison to chemotherapyalone. In this sense, analysis is needed of whetheridiotypic vaccination can produce not only long-lastingand complete remission, but even cure. It would be ofgreat interest to consider an optimisation of the experimentaldesign of clinical trials, an improvementof vaccine production, and the study of the molecularmechanisms of the tumour cell which modify the targetimmunoglobulin(AU)

[Past, present and future of anti-idiotype vaccination]. / Pasado, presente y futuro de la vacunación anti-idiotipo.

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.

[Active immunotherapy in the treatment of haematological neoplasias]. / Inmunoterapia activa en el tratamiento de neoplasias hematológicas.

The continuous search for therapeutic approaches that improve the conventional treatments of neoplasms, together with an improved understanding of the immune system, has led in recent years to the development of Immunotherapy. Basically, a distinction can be made between two forms of immunotherapy: passive immunotherapy, which consists in the transfer of antibodies or cells previously generated in vitro that are directed against the tumour, and active immunotherapy, which attempts to activate in vivo the immune system and induce it to elaborate a specific response against the tumor antibodies. Hematological neoplasms, specifically some B lymphomas, express in their membrane an immunoglobulin that is considered a specific antigen of the tumour, which is why these diseases have become the ideal target for immunotherapy treatments. There are many alternatives, ranging from protein vaccines, which have already shown clinical benefits, to those of the second generation, which make use of the new techniques of molecular biology to increase the efficacy of the vaccines and obtain their production in a quicker and less costly way, but with which there are not yet definitive clinical results.

Inmunoterapia activa en el tratamiento de neoplasias hematológicas / Active immunotherapy in the treatment of haematological neoplasias

La continua búsqueda de abordajes terapéuticos que mejoren los tratamientos convencionales de las enfermedades neoplásicas junto con el mejor conocimiento del sistema inmunitario ha llevado en los últimos años al desarrollo de la inmunoterapia. Básicamente se pueden distinguir dos formas de inmunoterapia: la inmunoterapia pasiva, que consiste en la transferencia de anticuerpos o células previamente generados in vitro que se dirigen contra el tumor, y la inmunoterapia activa, que pretende activar in vivo el sistema inmunitario e inducirlo a elaborar una respuesta específica contra los antígenos tumorales. Las neoplasias hematológicas, concretamente algunos linfomas B, expresan en su membrana una inmunoglobulina que se considera un verdadero antígeno específico de tumor; por eso estas enfermedades se han convertido en la diana ideal de los tratamientos de inmunoterapia. Las alternativas son muchas, desde las vacunas proteicas que ya han demostrado beneficios clínicos, hasta las de segunda generación, que aprovechan las nuevas técnicas de biología molecular para aumentar la eficacia de las vacunas y conseguir su producción de forma más rápida y menos costosa, pero con las que todavía no hay resultados clínicos definitivos (AU)

Characterization of an immunologically conserved epitope from hepatitis C virus E2 glycoprotein recognized by HLA-A2 restricted cytotoxic T lymphocytes.

BACKGROUND/AIMS: Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of importance because they can be used for vaccination, treatment of infection or monitoring of immune responses. Our purpose was to characterize new CTL epitopes in HCV structural proteins. METHODS: Peptides were synthesized and tested in HLA-A2 binding assays. Binder peptides were used to stimulate peripheral blood mononuclear cells from HCV+ patients and controls, and activity measured in chromium release and ELISPOT assays. RESULTS: Twenty binder peptides were found, and stimulation of HCV+ patient cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-A2. Peptide E2(614-622) was recognized by 30% of HLA-A2+ patients with chronic HCV infection, but no responses were observed in control groups. Five peptides derived from region E2(614-622) from 26 different viral isolates bound to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614-622) specific CTL. CONCLUSIONS: These results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.

Respuesta inmunitaria en la infección por el virus de la hepatitis C / Immune response in hepatitis C virus infection

La infección por el virus de la hepatitis C (VHC) se caracteriza por la alta tasa de cronificación viral. Aunque los tratamientos antivirales son eficaces en algunos pacientes, todavía queda un porcentaje alto de ellos que permanece crónicamente infectado. El estudio de los mecanismos del sistema inmunitario involucrados en la eliminación viral es importante tanto para el desarrollo de vacunas como para la comprensión de los fenómenos inmunopatológicos asociados a la infección. Aunque todos los pacientes con infección crónica desarrollan anticuerpos f rente a las proteínas virales, el alto grado de mutación de las regiones reconocidas por anticuerpos neutralizantes hace que la respuesta humoral no sea eficaz. Por otro lado, la eliminación de la infección por VHC tras una hepatitis aguda o tras el tratamiento con IFN- , se asocia con una potente respuesta de los linfocitos T CD4 y CD8, junto con un perfil de citocinas Th1. Sin embargo, la infección crónica viene acompañada con bajas respuestas inmunitarias c e l u l a res, así como un perfil de citocinas de tipo Th2.Además de su alta tasa de mutación, el VHC ha desarrollado mecanismos de interacción con componentes celulare s que desempeñan el normal funcionamiento del sistema inmunitario, permitiendo así la evasión de la respuesta y la cronificación viral. Las futuras vacunas, así como los tratamientos frente al VHC, habrán de tener en cuenta estos factores para conseguir una eliminación eficaz de la infección (AU)

Different doses of adenoviral vector expressing IL-12 enhance or depress the immune response to a coadministered antigen: the role of nitric oxide.

Joint immunization with two recombinant adenoviruses, one expressing hepatitis C virus (HCV) core and E1 proteins and another expressing IL-12 (RAdIL-12), strongly potentiates cellular immune response against HCV Ags in BALB/c mice when RAdIL-12 was used at doses of 1 x 105-1 x 107 plaque-forming units. However, cellular immunity against HCV Ags was abolished when higher doses (1 x 108 plaque-forming units) of RAdIL-12 were used. This immunosuppressive effect was associated with marked elevation of IFN-gamma and nitric oxide in the serum and increased cell apoptosis in the spleen. Administration of N-nitro-L -arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, to mice that received high doses of RAdIL-12 was lethal, whereas no apparent systemic toxicity by L -NAME was observed in those immunized with lower doses of the adenovirus. Interestingly, in mice immunized with recombinant adenovirus expressing core and E1 proteins of HCV in combination with RAdIL-12 at low doses (1 x 107 plaque-forming units), L -NAME inhibited T cell proliferation and CTL activity in response to HCV Ags and also production of Abs against adenoviral proteins. In conclusion, gene transfer of IL-12 can increase or abolish cell immunity against an Ag depending of the dose of the vector expressing the cytokine. IL-12 stimulates the synthesis of NO which is needed for the immunostimulating effects of IL-12, but apoptosis of T cells and immunosuppression ensues when IFN-gamma and NO are generated at very high concentrations.