DCs play a pivotal role in orchestrating innate and adaptive antitumor immunity. Activated DCs can produce large amounts of various proinflammatory cytokines, initiate T-cell responses, and exhibit direct cytotoxicity against tumor cells. They also efficiently enhance the antitumoral properties of NK cells and T lymphocytes. Based on these capabilities, immunogenic DCs promote tumor elimination and are associated with improved survival of patients. Furthermore, they can essentially contribute to the clinical efficacy of immunotherapeutic strategies for cancer patients. However, depending on their intrinsic properties and the tumor microenvironment, DCs can be rendered dysfunctional and mediate tolerance by producing immunosuppressive cytokines and activating Treg cells. Such tolerogenic DCs can foster tumor progression and are linked to poor prognosis of patients. Here, we focus on recent studies exploring the phenotype, functional orientation, and clinical relevance of tumor-infiltrating conventional DC1, conventional DC2, plasmacytoid DCs, and monocyte-derived DCs in translational and clinical settings. In addition, recent findings demonstrating the influence of DCs on the efficacy of immunotherapeutic strategies are summarized.

<p dir="ltr">Neutrophils are critical for innate immunity, acting as the body's first line of defense. They are terminally differentiated and are short-lived white blood cells. Cyclin-dependent kinases (CDKs), traditionally associated with cell cycle progression are now known to regulate crucial neutrophil functions: CDK2 influences neutrophil migration, CDK4 and 6 regulate neutrophil extracellular traps (NETs) formation, CDK5 controls degranulation, and CDK7 and 9 are pivotal for apoptosis and inflammation resolution.</p><p dir="ltr">Despite extensive studies on CDK2 in cell cycle regulation, its role in neutrophil function remained uncharacterized until recently. Inhibiting CDK2 kinase activity significantly impairs neutrophil migration. Using phosphoproteomic methods, we identified key proteins in multiple cellular pathways affected by CDK2 inhibition, with Cyclin D3 emerging as a binding partner. Direct substrates of CDK2, including RCSD1, CCDC6, LMNB1, and STK10, were found to be essential for neutrophil motility. These findings provide insights into the molecular mechanisms underlying this process. Consequently, targeting CDK2 or its substrates presents potential therapeutic strategies for conditions involving aberrant neutrophil migration or neutrophil-mediated inflammation, offering new avenues for treating neutrophil-dominant inflammatory diseases and advancing our understanding of neutrophil regulation.</p><p dir="ltr">Emergency granulopoiesis, a response to severe inflammation, involves the increased production of neutrophils in hematopoietic tissue. Understanding the body's response to severe inflammation necessitates more precise and less invasive methods to track neutrophil development. To distinguish newly formed neutrophils from existing ones in the occurrence of emergency granulopoiesis, we developed a transgenic zebrafish line expressing a time-dependent GFP-RFP switch fluorescent protein, enabling quantification through simple GFP/RFP ratiometric imaging. This method bypasses the limitations of traditional photo-labeling, which requires strong laser lines and label subsets of existing neutrophils.</p>

<p dir="ltr">Over the last century, cancer immunotherapy has become an attractive field due to the popularity of checkpoint blockades and adoptive cell therapy. Though these new frontier therapeutics are effective for certain populations, they’ve had either adverse effects on others or are non-efficacious when used to treat “cold tumors”. Hence, newer strategies are needed to sensitize cold tumors into immune-responsive “hot tumors”, which synergize with checkpoint blockades. The cyclic GMP-AMP synthase-Stimulator of INterferon Genes (cGAS-STING) pathway has been identified as a pathway that can initiate T cell infiltration and turn cold tumors into hot tumors. Therefore, STING agonists have been identified as potential remedies that could help bend the curve to increase the survival rate of cancer patients if combined with anti-PD1 and anti-CTLA4 therapies.</p><p dir="ltr">2’3’-cGAMP is a master regulator of the innate immune system and is produced by cGAS upon cancer deregulation as well as bacterial and viral infection. Although 2’3’-cGAMP is a nanomolar affinity binder to STING and has vast immunostimulatory potential, it is plagued by several limitations that prevent its use in vivo. Most medicinal chemists have focused on making phosphorothioate derivatives which circumvents 2’3’-cGAMP’s limitations, but synthesizing these analogs presents a synthetic challenge. Also, these derivatives are commonly administered via intratumoral injection, which is not an attractive mode of delivery. This dissertation tries to address some of these challenges and provide a newer platform to develop CDN-based STING agonists.</p><p dir="ltr">We describe a novel class of phosphorothioester-linked cyclic dinucleotides (endo-S-CDNs) as excellent STING agonists. Showing through structural-activity relationship (SAR) which groups are tolerated or detrimental for STING binding and cellular activity. Also, determining that these 5’-phosphorothioester-linked CDNs are resistant to cleavage by clinically relevant phosphodiesterases (PDEs). Finally, we discuss how this novel class of CDNs is suitable for subcutaneous dosing to clear tumors in different mouse models.</p>

New diagnostic methods and novel therapeutic agents spawn additional and heterogeneous in-formation, leading to an increasingly complex decision-making process for optimal treatment of cancer. A great amount of information is collected in organ-specific multidisciplinary tumor boards (MDTBs). By considering the patient’s tumor properties, molecular pathological test re-sults, and comorbidities, the MDTB has to consent an evidence-based treatment decision. Im-munotherapies are increasingly important in today’s cancer treatment, resulting in detailed in-formation that influences the decision-making process. Clinical decision support systems can fa-cilitate a better understanding via processing of multiple datasets of oncological cases and mo-lecular genetic information, potentially fostering transparency and comprehensibility of available information, eventually leading to an optimum treatment decision for the individual patient. We constructed a digital patient model based on Bayesian networks to combine the relevant pa-tient-specific and molecular data with depended probabilities derived from pertinent studies and clinical guidelines to calculate treatment decisions in head and neck squamous cell carcinoma (HNSCC). In a validation analysis, the model can provide guidance within the growing subject of immunotherapy in HNSCC and, based on its ability to calculate reliable probabilities, facilitates estimation of suitable therapy options. We compared actual treatment decisions of 25 patients with the calculated recommendations of our model and found significant concordance (Cohen’s κ=0.505, p=0.009) and 84% accuracy.

[ES] El cáncer de pulmón no microcítico (CPNM) representa un 80% de los casos de cáncer de pulmón, siendo uno de los tipos de cáncer más frecuentes y mortales. El tratamiento con inmunoterapia ha mejorado significativamente el pronóstico de los pacientes en las últimas décadas. No obstante, no todos los pacientes responden al tratamiento, por lo que se necesitan nuevos biomarcadores para predecir qué pacientes se podrían beneficiar de la inmunoterapia. El principal objetivo de esta tesis es obtener nuevos biomarcadores no invasivos para pacientes de CPNM avanzado tratados con inmunoterapia. Se incluyeron 52 pacientes de CPNM en estadios avanzados tratados con anti-PD1 o anti-PD1 en combinación con quimioterapia (anti-PD1+CT) en primera línea. Se analizaron biomarcadores no invasivos en muestras de sangre periférica, obtenidas antes del tratamiento y en la primera evaluación de respuesta. Los biomarcadores analizados en este estudio fueron: i) parámetros hematológicos e inmunológicos, ii) expresión de genes inmunoreguladores en células mononucleares de sangre periférica (PBMCs), iii) repertorio de TCR-ß y iv) genotipo de HLA. También se analizaron 13 controles sanos, y se observó que los pacientes con CPNM presentaron menores niveles de expresión de genes relacionados con las células T. Además, los pacientes con CPNM tenían menor número de clones de TCR-ß. Se analizó el valor predictivo y pronóstico de los potenciales biomarcadores independientemente en pacientes tratados con anti-PD1 o anti-PD+CT. Se encontraron biomarcadores con valor pronóstico, bien en las muestras basales o en las muestras tomadas en la primera evaluación de respuesta. Al utilizar muestras no invasivas, también se pudo estudiar la dinámica de los biomarcadores a lo largo del tratamiento, observando que algunos cambios ocurrían de manera diferencial en pacientes respondedores o dependiendo del tratamiento. La integración de los datos de las variables analizadas ha resultado en una propuesta de un modelo multivariante capaz de predecir qué pacientes tendrán mejor pronóstico, en el subgrupo de pacientes tratados con anti-PD1. Además, se crearon dos inmunogramas no invasivos incluyendo los ratios de los biomarcadores entre muestras tomadas antes y durante el tratamiento. Estos modelos se realizaron específicamente para cada tipo de tratamiento, y podrían ser útiles para monitorizar la respuesta durante el tratamiento. Este estudio resalta el papel de la biopsia líquida como una herramienta no invasiva para analizar biomarcadores de forma integral que permiten caracterizar y monitorizar el estatus inmune en pacientes con CPNM tratados con inmunoterapia o quimioinmunoterapia. / [CA] El càncer de pulmó no microcític (CPNM) representa un 80% dels casos de càncer de pulmó, i és un dels tipus de càncer més freqüents i mortals. El tractament amb immunoteràpia ha millorat significativament el pronòstic dels pacients en les últimes dècades. Malgrat això, no tots el pacients responen, per la qual cosa es necessiten nous biomarcadors per predir què pacients es beneficiaran del tractament amb immunoteràpia. El principal objectiu d'aquesta tesi és obtindre nous biomarcadors no invasius per a pacients de CPNM avançat tractats amb immunoteràpia. Es van incloure 52 pacients de CPNM en estadis avançats tractats amb anti-PD1 o anti-PD1 en combinació amb quimioteràpia (anti-PD1+CT) en primera línia. Es van analitzar biomarcadors no invasius a partir de mostres de sang perifèrica, que es van obtindre abans del tractament i en la primera avaluació de resposta. Els potencials biomarcadors analitzats en aquest estudi van ser: i) paràmetres hematològics i immunològics, ii) expressió de gens immunoreguladors en cèl·lules mononuclears de sang perifèrica (PBMCs), iii) repertori de TCR-ß i iv) genotip d'HLA. També es van analitzar 13 controls sans, i es va observar que els pacients amb CPNM presentaven menors nivells d'expressió de gens relacionats amb les cèl·lules T. A més, els pacients amb CPNM tenien menor riquesa de repertori de TCR-ß. S'han analitzat el valor predictiu i pronòstic dels potencials biomarcadors independentment en pacients tractats amb anti-PD1 o anti-PD1+CT. S'han trobat biomarcadors amb valor pronòstic, bé en les mostres basals o en les mostres preses en la primera avaluació de resposta. Com s'han utilitzat mostres no invasives, també s'ha pogut analitzar la dinàmica dels biomarcadores al llarg del tractament, i s'han observat canvis específics de pacients responedors o del tipus de tractament. La integració de les variables analitzades ha resultat en una proposta d'un model multivariant capaç de predir quins pacients amb CPNM tindran millor pronòstic, en el subgrup de pacients tractats amb anti-PD1. També s'han fet dos immunograms no invasius incloent els ràtios dels biomarcadors entre mostres preses abans i durant el tractament. Aquests models son específics per a cada tipus de tractament, i podrien ser útils per a monitorar la resposta durant el tractament. Aquest estudi ressalta el paper de la biòpsia líquida com una eina no invasiva per a analitzar biomarcadors de forma integral que permeten caracteritzar i monitorar l'estatus immune en pacients amb CPNM tractats amb immunoteràpia o quimioimmunoteràpia. / [EN] Non-Small Cell Lung Cancer (NSCLC) represents 80% of lung cancer cases, being one of the most frequent and death causing cancers. Recently developed treatments with immunotherapy have improved patient prognosis. However, a significant number of patients do not respond to treatment, thus there is an urgent need for biomarkers to predict which patients will benefit from immunotherapy. The main objective of this thesis was to obtain novel non-invasive biomarkers for advanced-stage NSCLC patients treated with immunotherapy. This study included 52 advanced-stage NSCLC patients treated with Anti-PD1 or Anti-PD1 in combination with chemotherapy (Anti-PD1+CT) in the first line setting. Non-invasive biomarkers were analysed using peripheral blood samples, which were obtained before first cycle and at first response assessment. The potential biomarkers analysed in this study were: i) haematological and immunological parameters, ii) immune-related gene expression analysed on Peripheral Blood Mononuclear Cells (PBMCs), iii) TCR-ß repertoire, and iv) HLA genotype. 13 healthy subjects were also included in this study. NSCLC patients presented lower T cell related gene expression levels than controls. Furthermore, cancer patients had a lower number of unique TCR-ß clones. We have assessed the predictive and prognostic value of the analysed variables independently on patients treated with anti-PD1 or anti-PD1+CT. We found prognostic biomarkers that could be useful to identify patients who benefit from treatment. Since we used non-invasive samples, we also observed differences in immune-related biomarkers at first response assessment in patients responding to treatment. In addition, biomarker dynamics were useful to identify changes occurring throughout treatment. The integration of data from the analysed variables has resulted in a proposal of a multivariate model capable of predicting patients with improved outcomes to treatment with anti PD1 therapy. Moreover, we have developed two non-invasive inmunograms including the ratios of on- and pre-treatment samples, which could be useful to monitor patients throughout treatment. Altogether, this study highlights the role of non-invasive biomarkers to characterize and monitor immune status in NSCLC patients treated with immunotherapy or chemoimmunotherapy. / This Thesis was supported by the following grants: Fundación Científica Asociación Española Contra el Cáncer. PRDVA18015MORE; Centro de Investigación Biomédica en Red Cáncer. Project B16/12/00350 e Instituto de Salud Carlos III: PI18/00266 / Moreno Manuel, A. (2024). Non-Invasive Immunogram. A Multidimensional Approach to Characterize and Monitor Immune Status in Non-Small Cell Lung Cancer [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204490

[ES] La presente tesis doctoral titulada "New advanced anti-tumor therapies based on hybrid mesoporous nanodevices" se centra en el diseño, síntesis, caracterización y evaluación de nuevos nanodispositivos híbridos orgánico-inorgánicos. En concreto, se han desarrollado nanopartículas de sílice mesoporosas (MSNs) y nanopartículas Janus combinando MSNs con platino y con oro con el objetivo de mejorar los tratamientos anti-tumorales. El primer capítulo es una introducción general que incluye una visión global del contexto en el que se enmarca la investigación realizada. En particular, se incluye información básica sobre los diferentes tipos de nanopartículas empleados en esta tesis doctoral, así como la descripción de la enfermedad del cáncer y la aplicación de los nanomateriales como terapia. A continuación, en el segundo capítulo, se presentan los objetivos generales y específicos de esta tesis doctoral. Los capítulos tercero y cuarto describen dos estrategias terapéuticas basadas en el desarrollo de nanopartículas con movimiento para mejorar la terapia antitumoral. Concretamente, en el primer capítulo experimental se presenta un nanodispositivo autopropulsado para la liberación controlada de fármacos en respuesta al glutatión (GSH) intracelular. Éste se basa en nanopartículas tipo Janus compuestas por sílice mesoporosa y oro, funcionalizadas en la parte del oro con la enzima catalasa, cargadas con doxorrubicina y con cadenas de oligo(etilenglicol) (S-S-PEG) unidas por puentes disulfuro en la cara de la sílice. Una vez sintetizado y caracterizado el dispositivo, se confirmó su movimiento y se demostró el funcionamiento de la puerta molecular. Finalmente, la internalización celular y la liberación de doxorrubicina se estudiaron en cultivos celulares. Motivados por los resultados anteriores, en el cuarto capítulo se describe un nuevo nanomotor diseñado como tratamiento antitumoral y similar al anterior. En este caso, la nanopartícula Janus desarrollada está compuesta por una nanopartícula de sílice mesoporosa junto con una de platino, cargada con doxorrubicina y cubierta con S-S-PEG. Al igual que en el trabajo anterior, se consigue la autopropulsión del dispositivo. Además, se obtuvo el perfil cinético de liberación del cargo en respuesta a estímulos y se confirmó su aplicación en cultivos celulares. Los capítulos cinco y seis se centran en una estrategia terapéutica nueva que consiste en potenciar la acción del sistema inmune sobre el tumor para conseguir su eliminación. En el primero de estos capítulos experimentales, se utilizaron nanopartículas MSNs cargadas con el fármaco JQ-1 y un siRNA frente al factor de crecimiento transformante ßeta (TGF-ß) como inmunoterapia. Una vez sintetizadas y caracterizadas, se confirmó la capacidad de estas nanopartículas para llevar a cabo la liberación de los cargos, junto con la disminución en la expresión de PD-L1 y en la producción de TGF-ß. Por último, se confirmó su aplicabilidad al ser capaces de inducir la eliminación de células de melanoma por el sistema inmune. De acuerdo con el último capítulo experimental, se describe un nuevo enfoque inmunoterapéutico basado en la comunicación química. En este caso, empleamos una nanopartícula Janus de oro y sílice funcionalizada con un péptido llamado pHLIP en la cara de sílice y el anticuerpo contra el receptor PD-1 unido a la cara de oro (J-pHLIP-PD1). La membrana de la célula tumoral es decorada por este nanodispositivo y gracias a la exposición del anticuerpo PD-1, se consiguió atrapar a los linfocitos T circulantes, desencadenando la eliminación de células tumorales por el sistema inmunitario. Además, estos resultados se confirmaron en un modelo metastásico B16-F10-Luc con una reducción de nódulos metastáticos. Finalmente, en el capítulo séptimo y octavo, se aborda la discusión general y las conclusiones derivadas de los estudios experimentales presentados en esta tesis doctoral. / [CA] Aquesta tesi doctoral titulada "New advanced anti-tumor therapies based on hybrid mesoporous nanodevices" se centra en el disseny, síntesi, caracterització i avaluació de nous nanodispositius híbrids orgànic-inorgànics. En concret, s'han desenvolupat nanopartícules de sílice mesoporoses (MSNs) i nanopartícules Janus combinant MSNs amb platí i amb or per al tractament antitumoral. El primer capítol és una introducció general que inclou una visió global del context on s'emmarca la recerca realitzada. En particular, es presenta informació bàsica sobre les nanopartícules emprades en aquesta tesi doctoral, així com la descripció de la malaltia del càncer i l'aplicació dels nanomaterials com a teràpia. A continuació, al segon capítol, es presenten els objectius generals i específics d'aquesta tesi doctoral. Els capítols tercer i quart descriuen dues estratègies terapèutiques basades en el desenvolupmanet de nanopartícules amb moviment per aconseguir una millora de la terapia antitumoral. Concretament, al primer capítol experimental es presenta un nanodispositiu autopropulsat per a l'alliberament controlat de fàrmacs en resposta al glutatió (GSH) intracel·lular. Aquest es basa en nanopartícules tipus Janus compostes per sílice mesoporosa i or, funcionalitzades a la part de l'or amb l'enzim catalasa, carregades amb doxorrubicina i amb cadenes d'oligo(etilenglicol) (SS-PEG) unides per ponts disulfur a la cara de la sílice. Una vegada sintetitzat i caracteritzat el dispositiu, es va confirmar la seua capacitat de moviment i es va demostrar el funcionament correcte de la porta molecular. Finalment, la internalització cel·lular i l'alliberament de doxorrubicina es van estudiar en cultius cel·lulars. Motivats pels resultats anteriors, al quart capítol es descriu un nanomotor nou dissenyat com a tractament antitumoral i similar a l'anterior. En aquest cas, la nanopartícula Janus desenvolupada està composta per una nanopartícula de sílice mesoporosa juntament amb una de platí, carregada amb doxorrubicina i coberta amb S-S-PEG. Igual que en el treball anterior, s'aconsegueix autopropulsió del dispositiu. A més, es va obtenir el perfil cinètic d'alliberament del càrrec en resposta a estímuls i se'n va confirmar l'aplicació en cultius cel·lulars. Els capítols cinc i sis se centren en una estratègia terapèutica nova que consisteix a potenciar l'acció del sistema immune sobre el tumor per aconseguir-ne l'eliminació. Al primer d'aquests capítols experimentals, es van utilitzar nanopartícules MSNs carregades amb el fàrmac JQ-1 i un siRNA davant del factor de creixement transformant ßeta (TGF-ß) com a immunoteràpia. Un cop sintetitzades i caracteritzades, es va confirmar la capacitat d'aquestes nanopartícules per dur a terme l'alliberament dels càrrecs, juntament amb la disminució de l'expressió de PD-L1 i de la producció de TGF-ß. Finalment, se'n va confirmar l'aplicabilitat en ser capaços d'induir l'eliminació de cèl·lules de melanoma pel sistema immune. D'acord amb el darrer capítol experimental, es descriu un nou enfocament immunoterapèutic basat en la comunicació química. En aquest cas, fem servir una nanopartícula Janus d'or i sílice funcionalitzada amb un pèptid anomenat pHLIP a la cara de sílice i l'anticòs contra el receptor PD-1 unit a la cara d'or (J-pHLIP-PD1). La membrana de la cèl·lula tumoral és decorada per aquest nanodispositiu i gràcies a l'exposició de l'anticòs PD-1, es va aconseguir atrapar els limfòcits T circulants, desencadenant l'eliminació de cèl·lules tumorals pel sistema immunitari. A més, aquests resultats es van confirmar en un model metastàtic B16-F10-Luc amb una reducció de nòduls metastàtics. Finalment, al capítol setè i vuitè, s'aborda la discussió general i les conclusions derivades dels estudis experimentals presentats en aquesta tesi doctoral. / [EN] The present PhD thesis entitled "New advanced anti-tumor therapies based on hybrid mesoporous nanodevices" focuses on the design, synthesis, characterization, and evaluation of new hybrid organic-inorganic nanodevices. We have developed mesoporous silica nanoparticles (MSNs) and Janus platinum-MSN and gold-MSN nanoparticles for tumor treatment. The first chapter is a general introduction that includes an overview of the context related to the research developed in this thesis. In particular, it includes basic information about different nanoparticles used in this doctoral thesis along with the description of cancer disease characteristics and the application of nanomaterials as therapy. Next, in the second chapter, the general and specific objectives of this Ph.D. thesis are presented. The third and fourth chapters describe two nanotechnology-based therapeutic strategies based on the development of nanomotors to improve cancer therapy. Specifically, the first experimental chapter presents a self-moving nanodevice for controlled drug release in response to intracellular glutathione (GSH). It is based on Janus gold-mesoporous silica nanoparticles functionalized with the enzyme catalase in the gold face, loaded with doxorubicin and capped with disulfide-linked oligo(ethylene glycol) (S-S-PEG) chains on the silica face. Once synthesized and characterized, the nanosystem motion was confirmed and the proper gating mechanism of the nanodevice was proven. Finally, the cellular uptake and doxorubicin release capacity have been demonstrated in cell cultures. Encouraged by the above results, chapter four describes a similar nanomotor design for antitumor therapy. In this case, the nanoparticle developed is composed of a Janus platinum-mesoporous silica nanoparticle, loaded with doxorubicin, and capped with S-S-PEG. As well as in the previous work, self-propulsion of the nanoparticles was achieved. Moreover, the stimuli-responsive cargo release kinetic profile was obtained and its application was confirmed in cell cultures. Chapters five and six focus on a new therapeutic strategy, empowering the immune system action on tumors to reach tumor cell death. In the first of these experimental chapters, JQ-1 and transforming growth factor-beta (TGF-ß) siRNA-loaded nanoparticles were used as efficient tumor immunotherapy. Once synthesized and characterized, the efficient cargo delivery was accomplished along with the programmed death-ligand 1 (PD-L1) downregulation and TGF-ß silencing. Lastly, its application was confirmed by triggering a specific immunogenic removal of tumor cells in melanoma cells. In chapter six, the development of a new communication-based immunotherapeutic approach is reported. In this case, we employ Janus gold-MSN functionalized with a peptide called pHLIP onto silica face and anti-PD-1 antibody bound to gold face (J-pHLIP-PD1). Tumor cell membrane is decorated by this nanodevice, leaving exposed on the surface PD-1 antibody which catches circulating T lymphocytes. It triggers immune system-induced-tumor leveling. Moreover, J-pHLIP-PD1 treatment-associated reduction of metastatic burden was also proven. Finally, in the seventh and eighth chapter, the general discussion and conclusions derived from the presented experimental studies of this Ph.D. thesis are exposed. / The authors thank the Spanish Government (Projects MAT2015-64139-C4-1, AGL2015-70235-C2-2-R, CTQ2014-58989-P, CTQ2015-71936-REDT and CTQ2017-87954-P) and the Generalitat Valencia (PROMETEO/2018/024) for support. The Comunidad de Madrid (IND2017/BMD-7642) is also gratefully acknowledged. The authors thank the Spanish Government (project PID2021-126304OB-C41, (MCUI/FEDER, EU)) and the Generalitat Valenciana (project PROMETEO CIPROM/2021/007). E.L-S is grateful for her FPU fellowship funded by MINECO (FPU18/06539). This work was supported by the European Research Council (ERC) via Advanced Grant (101052997, EDISON). This study forms part of the Advanced Materials program (MFA/2022/049) and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana. This work was supported by the European Research Council (ERC) via Advanced Grant (101052997, EDISON) and by CIBER -Consorcio Centro de Investigación Biomédica en Red- (CB06/01/2012), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación. / Lucena Sánchez, E. (2024). New advanced anti-tumor therapies based on hybrid mesoporous nanodevices [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/205528

L’avènement des inhibiteurs du point de contrôle immunitaire (ICIs) ciblant l’axe PD-1/PD-L1 a révolutionné le traitement des patients avec un carcinome pulmonaire non à petites cellules (CPNPC). Ce mémoire consolide les conclusions de trois études distinctes visant à analyser et à améliorer l'efficacité des ICIs en monothérapie chez le CPNPC. La première section explore les bénéfices associés au durvalumab suivant une chimioradiothérapie chez les patients présentant un CPNPC localement avancé, confirmant le bénéfice de survie associé aux ICI et élargissant les perspectives émises depuis l'étude PACIFIC, y compris au niveau de la valeur prédictive du PD-L1. Dans l’optique de caractériser de nouveaux biomarqueurs d’efficacité, la deuxième section souligne le rôle crucial du microbiome intestinal dans la modulation de la réponse aux ICIs, spécifiquement au niveau de la dysbiose intestinale liée aux antibiotiques. La méta-analyse proposée confirme l’impact délétère des antibiotiques sur la survie des patients traités avec un ICI, tout particulièrement lorsque l’antibiothérapie précède l’inhibition du PD-1/PD-L1. Dans le domaine des stratégies immunomodulatrices émergentes, la troisième section explore l’impact de la cryoablation chez les patients présentant un CPNPC avec PD-L1≥50% et traités avec le pembrolizumab. Sans relever un signal d’efficacité supérieure, cette étude de phase I/II confirme la faisabilité et l’innocuité de la cryoablation, une technique permettant la libération d’antigènes tumoraux en circulation sans dénaturation thermique. Ultimement, ce mémoire propose un survol des bénéfices de survie, biomarqueurs prédictifs et stratégies immunomodulatrices liés à l’utilisation des ICIs chez les patients avec un CPNPC avec l’espoir d’optimiser les paradigmes thérapeutiques existants. / The advent of immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis has revolutionized the therapeutic landscape for patients diagnosed with non-small cell lung cancer (NSCLC). This thesis consolidates the findings of three distinct studies aiming to analyze and enhance the efficacy of ICI monotherapy in NSCLC. The first section delves into the real-world use of durvalumab following chemoradiotherapy in stage III NSCLC, confirming the survival benefit associated with ICI administration in this context and broadening the insights derived from the PACIFIC study, particularly regarding the predictive value of PD-L1. With the aim of characterizing new biomarkers of efficacy, the second section sheds light on the crucial role of the gut microbiome in modulating responses to ICIs, particularly intestinal dysbiosis related to antibiotics. The meta-analysis confirms the detrimental impact of antibiotics on the overall survival of patients with advanced cancer treated with ICI monotherapy, especially when antibiotic therapy precedes PD-1/PD-L1 inhibition. In the realm of emerging immunomodulatory strategies, the third section explores the impact of cryoablation in patients with NSCLC and PD-L1≥50% treated with pembrolizumab. Although the procedure did not translate into a signal of superior efficacy, the proposed phase I/II study confirms the feasibility and safety of cryoablation, a technique allowing the release of circulating tumor antigens without heat-related denaturation. Ultimately, this thesis presents a contemporary overview of survival benefits, predictive biomarkers, and immunomodulatory strategies associated with the use of ICIs in monotherapy in patients with NSCLC, with the hope of optimizing existing therapeutic paradigms.

Die adoptive T-Zell-Therapie (ATT) ist eine sich schnell entwickelnde Immuntherapie, die bei Patienten, die an verschiedenen Krebsarten leiden, eine positive klinische Reaktion anzeigt. Eine Variante der ATT ist eine T-Zellen-Rezeptor (TCR)-Gentherapie, bei der Patienten-T-Zellen mit krebsspezifischen TCRs ausgestattet werden. Die Herstellung der TCR-erzeugten T-Zellen ist schnell und robust und erfordert eine geringe Anfangsmenge an Patienten-T-Zellen. Der Mangel an verfügbaren krebsspezifischen TCRs, die auf verschiedene Moleküle des menschlichen Leukozytenantigens (HLA) der Klasse I beschränkt sind, schließt jedoch viele Patienten von der Krebsbehandlung aus. Die Generierung einer krebsspezifischen TCR-Bibliothek, die aus gut definierten TCRs besteht, könnte die Zahl der Patienten, die an klinischen Studien teilnehmen, erhöhen. Das Ziel dieser Doktorarbeit war es, Epstein-Barr-Virus (EBV)-spezifische TCRs zu identifizieren und zu isolieren, um eine EBV-spezifische TCR-Bibliothek als ein nützliches Werkzeug der TCR-Gentherapie bei der Behandlung von EBV-bedingten Krebserkrankungen zu generieren. Insgesamt wurden neun EBV-spezifische TCRs von EBV-positiven Spendern isoliert und charakterisiert, die verschiedene pHLA-Komplexe von EBV-Latentmembranproteinen (LMP1, LMP2A) und Kernprotein (EBNA3C) erkannten. Zusätzlich wurde ein neuartiges immunogenes LMP1-Epitop (QQNWWTLLV) entdeckt, das auf HLA-C*15:02 beschränkt ist. Definierte EBV-spezifische TCRs können als Grundlage für die EBV-spezifische TCR-Bibliothek verwendet werden, die eine wertvolle Quelle von TCRs für die schnelle Generierung von EBV-spezifischen T-Zellen zur Behandlung von Krebspatienten mit verschiedenen HLA-Typen darstellt. / Adoptive T cell therapy (ATT) is a fast developing immunotherapy indicating positive clinical response in patients suffering from different type of cancers. One type of the ATT is a T cell receptor (TCR) gene therapy, which involves endowing patient T cells with cancer-specific TCRs. Manufacturing of the TCR-engineered T cells is fast and robust, requiring small initial amount of patient T cells. However, lack of available cancer-specific TCRs restricted to various human leukocyte antigen (HLA) class I molecules eliminates many patients from cancer treatment. Generation of a cancer-specific TCR library consisting of well-defined TCRs could increase the number of patients enrolled in clinical trials. The aim of this PhD thesis was to identify and isolate Epstein-Barr virus (EBV)-specific TCRs in order to generate the EBV-specific TCR library as a useful tool of the TCR gene therapy for treatment of EBV-related malignancies. In total, nine EBV-specific TCRs of EBV-positive donors that recognized various pHLA complexes of EBV latent membrane proteins (LMP1, LMP2A) and nuclear protein (EBNA3C) were isolated and characterized. Additionally, a novel immunogenic LMP1 epitope (QQNWWTLLV) restricted to a HLA-C*15:02 was discovered. Defined EBV-specific TCRs can be used as a basis for the EBV-specific TCR library, which provides a valuable source of TCRs for rapid generation of EBV-specific T cells to treat cancer patients with different HLA types.

Das Induzieren und Aufrechterhalten einer tumor-protektiven Immunität sind wesentliche Ziele in der Immuntherapie des Neuroblastoms. Eine Erhöhung der Anzahl von tumor-infiltrierenden Leukozyten könnte ein Weg sein, um dieses Ziel zu erreichen. Fractalkine ist ein besonderes TH1 CX3C Chemokin, welches sowohl Adhäsion und Migration von Leukozyten vermittelt. Gerichtetes IL-2 (ch14.18-IL-2) wurde durch eine genetische Fusion von anti-GD2 Antikörper mit IL-2 hergestellt, damit IL-2 spezifisch in das Mikromilieu von Neuroblastomen gebracht werden kann. In dieser Arbeit habe ich die Hypothese getestet, dass Gentherapie mit dem Chemokin Fractalkine (FKN) eine wirksame Antineuroblastom-Immunantwort induziert, welche durch gerichtetes IL-2 amplifiziert wird. Zu diesem Zweck wurden NXS2-Zellen genetisch verändert, damit sie murines FKN produzieren (NXS2-FKN). Transkription und Expression des mFKN Gens konnte in NXS2-FKN Zellen und Tumorgewebe gezeigt werden. Die chemotaktische Eigenschaft von FKN wurde sowohl in vitro als auch in vivo gezeigt. FKN zeigte eine Reduktion des Primärtumorwachstums, welches durch gerichtetes IL-2 mit nicht-kurativen Dosen von ch14.18-IL-2 deutlich verbessert wurde. Ferner wurden experimentelle Lebermetastasen nur in den Mäusen komplett eradiziert, welche die Kombinationstherapie erhalten haben. Die Mechanismen, welche an dieser Antitumorantwort beteiligt sind, schließen eine wirksame T-Zell-Aktivierung (Hochregulation von CD69, CD25, und von TNF-alpha und INF-gamma), sowie eine Erhöhung der tumorspezifischen CTL-Aktivität mitein. Die Depletion von CD4+ und CD8+ T-Zellen in vivo hat diesen therapeutischen Effekt aufgehoben, was die essentielle Rolle von T-Zellen in diesem immuntherapeutischen Ansatz unterstreicht. Zusammenfassend konnte ich zum ersten Mal zeigen, dass Chemokin-Gentherapie mit FKN durch gerichtetes IL-2 amplifiziert wird, was eine Kombination dieser beiden Strategien zur adjuvanten Therapie beim Neuroblastom nahe legt. / Induction and maintenance of tumor-protective immunity are the major goals of neuroblastoma immunotherapy. Enhancing the amount of tumor infiltrating leukocytes might be a way to achieve these goals since they may be associated with residual evidence of the ineffective immune response. Fractalkine is a unique TH1 CX3C chemokine known to induce both adhesion and migration of leukocytes mediated by a membrane-bound and a soluble form, respectively. Targeted IL-2 (ch14.18-IL-2) was constructed by anti-GD2 antibody fused with IL-2 so that IL-2 can be directed into the microenvironment of neuroblastoma tumor. Here, I tested the hypothesis that chemokine gene therapy with fractalkine (FKN) induces an effective anti-neuroblastoma immune response amplified by targeted IL-2. NXS2 cells were engineered to stably produce murine FKN (NXS2-FKN). Transcrip- tion and expression of the mFKN gene in NXS2-FKN cells and tumor tissue were demonstrated. The chemotactic activity of FKN expressed by NXS2 cells was determined both in vitro and in vivo. Importantly, NXS2-FKN exhibited a reduction in primary tumor growth, which was boosted by targeted IL-2 using non-curative doses of ch14.18-IL-2. Furthermore, experimental liver metastases were completely eradicated in mice receiving the combination therapy, demonstrating the induction of a long-lived tumor protective response. The mechanisms involved in antitumor response included effective T cell activation as indicated by the up-regulation of T-cell activation markers (CD69, CD25) and proinflammatory cytokines (TNF-alpha, INF-gamma) as well as the enhancement of tumor specific CTL activity. The depletion of CD4+ and CD8+ T cells in vivo abrogated the therapeutic effect supporting the crucial role of T cells in this immunotherapeutic approach. In summary, I demonstrated for the first time that chemokine gene therapy with FKN is amplified by targeted IL-2 suggesting a combination of both strategies as an adjuvant therapy for neuroblastoma.

Die spezifische adoptive Immuntherapie ist ein hoffnungsvoller Ansatz zur Behandlung von Tumoren. Die aufwendige individuelle Bereitstellung primärer Effektorlymphozyten könnte durch den Einsatz etablierter tumorantigenspezifischer Effektorzellinien vermieden werden. In dieser Arbeit wurde untersucht, ob sich ein Tumortargeting der humanen Natürlichen Killer-(NK)-Zellinie YT durch den Gentransfer chimärer Immunglobulin-T-Zellrezeptoren (cIgTCRs) erreichen läßt. Die cIgTCR-Konstrukte wurden aus single-chain-Fv-Fragmenten (scFv), dem IgG1-Fc-Teil und der CD3-Zeta-Signalkette erzeugt. Die scFv-Fragmente wurden aus den humanisierten Antikörpern BW431/26 und HuM195, die spezifisch für das karzinoembryonale Antigen (CEA) bzw. CD33 sind, konstruiert und zeigten als scFv-hFc-Fusionsproteine eine spezifische Bindung an Tumorzellen. Die YT-Zellen wurden mit den cIgTCR-Genkonstrukten über Elektroporation transfiziert und über immunologische Verfahren angereichert. In-vitro-Studien ergaben eine spezifische Lyse von CEA+ Kolonkarzinomzellinien durch die scBW431/26-hFcZeta+ YT-Zellen. Die Zytotoxizität korrelierte mit der Expression des cIgTCR-Antigens auf den Tumorzellen und wurde durch zirkulierendes CEA nicht gehemmt. Die scHuM195-hFcZeta+ YT-Zellen zeigten eine spezifische Lyse der CD33+ myeloischen Leukämiezellinie KG1. Die Bestrahlung wurde zur Wachstumsbegrenzung der YT-Zellen eingesetzt. Die spezifische Zytotoxizität der scBW431/26-hFcZeta+ YT-Zellen gegenüber CEA+ Tumorzellen war einen Tag nach Bestrahlung unverändert. Die Koinjektion von CEA+ Tumorzellen mit bestrahlten scBW431/26-hFcZeta+ YT-Zellen führte zu einer signifikanten Hemmung des Tumorwachstums in NOD/SCID-Mäusen. Die cIgTCR+ YT-Zellen zeigten in vitro eine geringe Sensibilität gegenüber allogenen Blutlymphozyten. Die Ergebnisse zeigen, daß die Zytotoxizität der NK-Zellinie YT tumorantigenspezifisch durch cIgTCR-Gentransfer erweitert wird und ein Potential zur Behandlung minimaler Tumorerkrankungen besteht. / The specific adoptive immunotherapy is a promising strategy for cancer treatment. The utilization of established tumor antigen specific effector cell lines could bypass the expendable individual preparation and often limited specificity of primary effector lymphocytes. This study investigated the tumor targeting of the human Natural Killer (NK) cell line YT by gene transfer of chimeric immunoglobulin T cell receptors (cIgTCRs). The cIgTCR constructs were generated of single chain antibody fragments (scFv), the IgG1 Fc part and the CD3 Zeta chain. The scFv fragments were constructed of the humanized antibodies BW431/26 and HuM195 with specificity for the carcinoembryonic antigen (CEA) and CD33, respectively, and showed as scFv-Fc fusion proteins a specific binding to tumor cells. YT cells were transfected with the cIgTCR gene constructs by electroporation and enriched by immunological cell separation. In vitro studies revealed a specific lysis of CEA+ colon carcinoma cell lines by scBW431/26-hFcZeta+ YT cells. The cytotoxicity correlated with the expression of the cIgTCR antigen on the tumor cells and was not inhibited in the presence of soluble CEA. The scHuM195-hFcZeta+ YT cells mediated a specific lysis of the CD33+ myeloic leukemia cell line KG1. The irradiation was used to limit the growth of the YT cell line. The specific cytotoxicity of the scBW431/26-hFcZeta+ YT cells against CEA+ tumor cells was unaltered one day after irradiation. The coinjection of CEA+ tumor cells and irradiated scBW431/26-hFcZeta+ YT cells led to a significant growth inhibition in NOD/SCID mice. The cIgTCR+ YT cells showed a low susceptibility to the cytotoxicity of allogeneic blood lymphocytes in vitro. The results demonstrated that the cytotoxicity of the human NK cell line YT can be specifically extended to tumor antigens by cIgTCR gene transfer. The employment of receptor gene modified YT cells could be a useful tool for the adoptive immunotherapy of minimal tumor diseases.