IX Forum on Translational Immunology and Cancer Immuno-therapy (FIT Cancer 9) ONCOIMMUNOLOGY
Immunotherapy,cancer,tumor immunology
ABSTRACT/INTRODUCTION Tumor immunology and cancer immunotherapy is a growing field, continuously evolving. Different types of immunotherapies have been implemented over last decade in the standard of care across several tumor types. Novel strategies to overcome resistance or to tackle the tumor microenvironment are currently at the forefront of the tumor immunology and cancer immunotherapies research. The Spanish Group for Cancer Immuno-Biotherapies (GÉTICA) held the IX Forum on Translational Immunology and Cancer Immunotherapy (FITCancer 9) from 9-11 March, in Madrid (Spain). FITCancer, which is the largest meeting uniquely focused on cancer immunotherapy, brings together clinicians and researchers, experts in the field of cancer immunology and immunotherapy. Here, we present abstracts submitted by GÉTICA’s members to the IX Forum on Translational Immunology and Cancer Immunotherapy.
IX Forum on Translational Immunology and Cancer Immuno-therapy (FIT Cancer 9) ONCOIMMUNOLOGY

Tumor immunology and cancer immunotherapy is a growing field, continuously evolving. Different types of immunotherapies have been implemented over last decade in the standard of care across several tumor types. Novel strategies to overcome resistance or to tackle the tumor microenvironment are currently at the forefront of the tumor immunology and cancer immunotherapies research. The Spanish Group for Cancer Immuno-Biotherapies (GÉTICA) held the IX Forum on Translational Immunology and Cancer Immunotherapy (FITCancer 9) from 9-11 March, in Madrid (Spain). FITCancer, which is the largest meeting uniquely focused on cancer immunotherapy, brings together clinicians and researchers, experts in the field of cancer immunology and immunotherapy. Here, we present abstracts submitted by GÉTICA’s members to the IX Forum on Translational Immunology and Cancer Immunotherapy.

Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting anti-CD1a T cell engagers Anaïs Jiménez-Reinosoa; Néstor Tiradob; Alba Martínez-Morenob; Víctor M. Díazc; Marina García-Peydród; Oana Hangiua; Laura Díez-Alonsoa; Ángela Albitred; Petronila Penelad; María L. Toribiod; Pablo Menéndeze; Luis Álvarez-Vallinaa; Diego Sánchez Martínezb aCancer Immunotherapy Unit (UNICA). Department of Immunology. Hospital Universitario 12 de Octu7bre. Madrid. Immuno-Oncology and Immunotherapy Group. Instituto de Investigación Sanitaria 12 de Octubre (imas12). Madrid. bJosep Carreras Leukemia Research Institute (IJC). Barcelona. cOneChain Immunotherapeutics S. L. Barcelona. dCentro de Biología Molecular Severo Ochoa CSIC-UAM. Madrid. Instituto de Investigación Sanitaria La Princesa. Madrid. eRed Española de Terapias Avanzadas (TERAV) – Instituto de Salud Carlos III (ISCII) (RICORS). Madrid. Centro de Investigación Biomédica en Red – Oncología (CIBERONC) T cell acute lymphoblastic leukemia (T-ALL) is a phenotypically and genetically heterogeneous malignant disorder which comprises ~15% and ~25% of all acute leukemias diagnosed in children and adults, respectively. Intensive chemotherapy regimens and hematopoietic cell transplantation have led to the improved survival of T-ALL patients, but the event-free and overall survival (OS) remains <70%, and relapsed/refractory (R/R) T-ALL has a particularly poor outcome. CD1a represents a safe target for cortical T-ALL (coT-ALL) patients, and fratricide-resistant CD1a-directed chimeric antigen receptor (CAR) T cells have been preclinically validated as an immunotherapeutic strategy for R/R coT-ALL. However, T-ALL relapses are commonly very aggressive and hyperleukocytic, posing a challenge to recover sufficient non-leukemic effector T cells from leukapheresis in R/R T-ALL patients. Through several in vitro and in vivo cutting-edge models of coT-ALL we aim to analyze the efficacy of engineered T cells secreting CD1a x CD3 T cell-engaging Antibodies (CD1a-STAb) in comparison to engineered T cells expressing a second-generation CD1a-CAR. In vitro binding, T cell activation, cytokine secretion, cytotoxicity and escape assays were conducted. The in vivo anti-leukemic effect of CD1a-STAb T cells was evaluated in a short-term xenograft and in two long-term patient-derived xenograft coT-ALL models. We showed that CD1a-T cell engagers bind to cell surface expressed CD1a and CD3 and induced specific T cell activation. In vitro cytotoxicity assays at different time points or effector:target (E:T) ratios demonstrated that STAb-CD1a T cells induce more potent and rapid cytotoxic responses than CAR-CD1a T cells. Moreover, recruitment of bystander T cells endowed CD1a-STAbs with an enhanced in vitro cytotoxicity than CD1a-CAR T cells at lower E:T ratios. In in vivo coT-ALL xenograft models, CD1a-STAb T cells demonstrated to be as effective as CD1a-CAR T cells. This study validates the efficacy of T cell redirecting strategies targeting CD1a for coT-ALL and supports the therapeutic use of CD1a-STAbs as alternative to CD1a-CAR T cells in coT-ALL patients with limited numbers of non-leukemic effector T cells. Funding PID2020-117323RB-100, PDC2021-121711-100, DTS20/00089, AECC-PROYE19084ALVA, FCRIS-2018-0042, FCRIS-2021-0090, LCF/PR/HR19/52160011, ERC-PoC-957466, PID2019-108160RB-I00, PID2019-105623RB-I00, AECC-CICPF18030TORI, PI21-01834, FPU19/00039, IND2020/BMD-17668, CM20/00004, PTQ2020-011056, CD19/00013. Circulating DNA and flow cytometry as an emerging liquid biopsy biomarker to predict the response to the immunotherapy in melanoma and lung cancer patients Elisabeth Pérez Ruiza,b; Juan Luis Onieva-Zafrab,c; Javier Oliverb,c; Miguel Ángel Berciano- Guerreroa; Patricia Chavesc; Rosa María Villatoro-Roldánd; Beatriz Martínezb; Alexandra Canteroa; María Garrido-Barrosb; Antonio Ruedaa,b; Isabel Barragan-Mallofretb,c,e aMedical Oncology Department, Regional and Virgen de la Victoria University Hospitals, Malaga, Spain. bBiomedical Research Institute of Malaga and BIONAND Nanomedicine Platform (IBIMA BIONAND Platform) Translational Research in Cancer Immunotherapy group, Malaga, Spain. cFaculty of Medicine, University of Malaga, Malaga, Spain. dMedical Oncology Department, Hospital Costa del Sol. eGroup of Pharmacoepigenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Numerous single biomarker analyses to predict immunotherapy (IT) response and prognosis have been proposed. However, only few have shown robust predictions and none of them has been prospectively validated. In this study, we have developed a multimarker score combining cellular and molecular blood analytes that allows us to stratify basally and early according to long-term response and to predict prognosis. A longitudinal prospective study including metastatic non-small lung cancer (NSCLC) and melanoma patients treated with IT was conducted for one year with basal and repeated on-treatment analyses on two independent cohorts: Discovery and Validation. The circulating analytical parameters tested for the score consisted of quantification of plasma cell-free DNA, CD3, CD8, PD1, PDL1, CD4, Tregs and NK cell populations measured by Flow-Cytometry, and the routine clinical quantification of neutrophiles, lymphocytes, leukocytes, hemoglobin, platelets, as well as the neutrophils to lymphocytes ratio. The predictive CircScore was calculated by a multivariant Cox regression model using a cutoff that allows the differentiation between Low (associated with clinical benefit) and High (associated with immunotherapy resistance). The model was evaluated as prognostic using Kaplan-Meier survival analyses with the Log-rank test, and the C-Index metric that evaluates the prognostic power of the model. A total of 90 (58 NSCLC and 32 melanoma) patients in the Discovery cohort and 40 patients in the Validation cohort were included. After one year of treatment, 30 patients were classified as good responders (33,3%) and 58 as bad responders to IT (66,7%). The resulting score combined T1-cfDNA, T1-NLR, T1-Tregs, T2-CD3, T2-CD4, and T2-Platelets. Those patients with a Low score had a much longer OS than those with a High score, and this was replicated in the Validation cohort (3.39 vs 33.8 months, p < .00001). Moreover, the CircScore was calculated independently in the melanoma and NSCLC patients and similar results were obtained. Finally, the C-Index evidenced the superiority of CircScore compared with a clinical model composed of sex, LDH, ECOG, and metastatic sites. CircScore is a clinically implementable biomarker combination score for basal and early stratification by response and that improves the prognosis prediction performance of some of the most established immunotherapy response biomarkers across different tumor types. Funding PI-0135-2018 Enhancing antitumor response by combining immune checkpoint blockade and direct anti-cancer action with a PD-L1/EGFR bispecific antibody Laura Rubio-Péreza; Rodrigo Lázaro-Gorinesa; Seandean L. Harwoodb; Marta Comptec; Rocío Navarroc; Antonio Tapia-Galisteoa; Jaume Bonetd; Belén Blancoa; Simon Lykkemarke; Ángel Ramírez-Fernándeza; Carmen Domínguez-Alonsoa; Laura Díez- Alonsoa; Alejandro Segura-Tudelaa; Oana Hangiua; Ainhoa Erce-Llamazaresa; Francisco J. Blancof; Cruz Santosg; José L. Rodríguez-Peraltoh; Laura Sanzi; Luis Álvarez-Vallinaa aCancer Immunotherapy Unit (UNICA). Department of Immunology. Hospital Universitario 12 de Octubre. Madrid, Spain. bDepartment of Molecular Biology and Genetics. Aarhus University. Denmark. cDepartment of Antibody Engineering. Leadartis SL. Tres Cantos, Madrid. Spain. dInstitute of Bioengineering. École Polytechnique Fédérale de Lausanne. Lausanne, Switzerland eImmunotherapy and Cell Engineering Laboratory. Department of Engineering. Aarhus University. Aarhus, Denmark. fCentro de Investigaciones Biológicas Margarita Salas (CIB). CSIC. Madrid, Spain. gFaculty of Experimental Sciences. Universidad Francisco de Vitoria (UFV). Pozuelo de Alarcón, Madrid. Spain. hDepartment of Pathology. Hospital Universitario 12 de Octubre. Madrid, Spain. iCentro de Investigación Biomédica en Red en Oncología (CIBERONC). Madrid, Spain Immune checkpoint blockade (ICB) with antibodies have shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. The design of new bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells is a novel strategy that could improve the efficacy of current immunotherapies. We developed a PD-L1/EGFR bsAb by fusing a dual-targeting tandem trimerbody with the human IgG1 hinge and Fc regions. The bsAb was characterized by structural analysis, in vitro functional studies, and antitumor efficacy in humanized mice bearing aggressive triple-negative breast cancer and lung cancer xenografts. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction and induce strong antigen-specific antibody dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were further observed. In addition, tumor growth control was associated with a significantly increased proportion of CD8 + T cells. These results support the development of IgTT-1E for the treatment of EGFR positive cancers. Funding PID2020-113225GB-I00, PID2020-117323RB-100, PI20/01030, PI19/00132, DTS20/00089, FCRIS-2021-0090), AECC PROYE19084ALVA, HR21-00761 project IL7R_LungCan. PRE2018-083445, CM20/00004, IND2020/BMD-17668, IFI18/00045. L.R-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/ Merck. Humoral and cellular immune responses induced by full academic anti-CD19 chimeric antigen receptor (ARI-0001 cells) Ariadna Bartoló-Ibarsa,b,e; E. Azucena González-Navarroa,b; Alexandra Johanssong; Timothy Kountzg; Nela Klein-Gonzáleza,b,e; Berta Casanovas-Albertí a,b,e; Valentín Ortiz-Maldonadoc; Marta Español-Rego a,b; Maria Castellà a,b; Daniel Benítez a,b; Raquel Cabezón a,b; Jordi Esteve c,e,f; Jordi Yagüe b,e; Mariona Pascal a,b; Álvaro Urbano-Ispizua c,e,f; Susana Rivesd; Julio Delgado c,e,f; Manel Juan a,b,e,f*; William W. Kwokg* a Unitat d’ Immunoteràpia, HSJD-Hospital Clínic Barcelona, Barcelona, Spain; b Immunologia, CDB, Hospital Clínic, Barcelona, Spain; c Hematologia, ICMHO, Hospital Clínic Barcelona, Barcelona, Spain; d Hematologia, Hospital Sant Joan de Déu (HSJD), Barcelona, Spain; e Institut d’Investigacions Biomèdiques August Pi i Sunyer IDIBAPS/FCRB Barcelona, Spain; f Universitat de Barcelona, Barcelona, Spain; g Benaroya Research Institute, Seattle, Washington *Both autors share co-seniorship of this work. ARI-0001 cells are indicated to treat patients with B-cell acute lymphoblastic leukemia older than 25 year old. This adoptive cellular therapy was approved by Spanish Agency of Medicine in 2021 under hospital exemption, next to four other anti-CD19 constructs (Kymriah Yescarta, Tecartus and Breyanzi). ARI-0001 cells express an anti-CD19 single chain variable fragment (scFv), derived from a murine monoclonal antibody, conjugated with the transmembrane and co-stimulatory domains. Even though safety and efficacy has been proven in the clinical trial CART-BE-01, it is still not fully investigated humoral and cellular immune responses induced by ARI-0001 cells. Regarding humoral immune responses, human anti-murine antibodies (HAMA) in patients’ sera have been detected with flow cytometry. Subsequently, cytotoxicity assays have been performed to assess HAMA neutralizing effect on ARI-0001 cells. Regarding cellular immunogenicity, possible immunogenic peptides were predicted in silico according to the HLA of treated patients. Focusing on HLA class II, a total of 35 prioritized as possible immunogenic peptides were used to stimulate PBMCs from treated patients and to sort CD4 + T- cells through activation induced markers, CD154 and CD69. After expansion, CD4 + T-cells were screened again with peptide pools and then individual peptides. Finally, HLA class II (DP, DQ and DR) was blocked to know which HLA present the peptide which triggers T-cell activation. Twenty out of 47 patients (42.5%) tested positive for the presence of anti-CAR antibodies and only 4 out of 20 patients (20%) had HAMA with neutralizing effect on ARI-0001 cells. Regarding cellular immune response, it has been obtained 43 cell lines from 5 different patients who developed human anti-murine antibodies. Eight out of 43 cell lines were reactive against 35 HLA-class II CAR19 peptides after one month of expansion. In relation to CAR19 peptides, immunogenic peptides for these eight cell lines were located at scFv and were presented by HLA-DR. All these results suggest that our academic construct induces humoral and cellular immune responses. Specifically, scFv seems to be the most immunogenic domain. Therefore, assessment of immunogenicity induced by ARI-0001 cells could be important, especially before considering a second infusion. POSTER SESSION Long-term outcomes of a prospective multicenter study on the combination of stereotactic ablative radiotherapy and anti-PD-1 in oligoprogressive non-small cell lung cancer and melanoma Juan Zafraa; Rodolfo Chicas-Settb; Andrés Verac; Delvys Rodríguezd; Juan Castillab; Juan Luis Onievaa; Laura Ferrerac; Bárbara Salasc; Marta Lloretc; Pedro C. Larae; Isabel Barragána aGrupo de Investigación Traslacional en Inmunoterapia del Cáncer. Centro de Investigaciones Médico-Sanitarias (CIMES). Universidad de Málaga (UMA). Málaga. Instituto de Investigación Biomédica de Málaga (IBIMA). Málaga. bServicio de Oncología Radioterápica. ASCIRES Grupo Biomédico. Valencia. cServicio de Oncología Radioterápica. Hospital Universitario de Gran Canaria Doctor Negrín. Las Palmas. dServicio de Oncología Médica. Hospital Universitario Insular de Gran Canaria. Las Palmas. eCentro Oncológico Integral Canario. Hospital Universitario San Roque. Las Palmas The number of patients with metastatic non-small cell lung cancer (NSCLC) and melanoma who benefit from anti−programmed cell death protein 1 (anti-PD-1) remains low due to resistance mechanisms. Stereotactic ablative radiotherapy (SABR) can improve responses to anti-PD-1 in oligoprogressive disease. We present extended outcomes, with a focus on long-term survivors, of a multicenter prospective observational study which aimed to determine whether concomitant anti-PD-1 and SABR to oligoprogressive sites can enhance tumor response in this setting. Patients with metastatic NSCLC or melanoma in progression to anti-PD-1 but continuing the same line owing to clinical benefit were referred for palliative SABR. All patients received concomitant pembrolizumab or nivolumab and SABR to 1 to 5 lesions, maintaining anti-PD-1 until further progression, unacceptable toxicity, or medical/patient decision. Objective response rate (ORR) was evaluated during all follow-up according to Response Evaluation Criteria in Solid Tumors 1.1. The abscopal response (AR) was evaluated 8 weeks after SABR as a ≥ 30% reduction in up to 2 predefined nonirradiated lesions. With a median follow-up of 51.8 months, ORR was 40% (32% complete responses and 8% partial responses) in the 50 patients included. Median progression-free survival was 17.7 months (95% CI, 6.9–32 months). Median overall survival (OS) since SABR was 34.6 months (95% CI, 20.2 months-not reached). Estimated five-year PFS and OS since SABR were 27.8% (95% CI, 16.9%-45.8%) and 36.1% (95% CI, 23.4%-55.7%), respectively. AR was 65%, evaluated in 40 patients who fulfilled the criteria. Twenty patients (40%) surpassed median OS and were considered long-term survivors (range, 36.8–65.1 months). Median PFS in this subgroup was 32 months (95% CI, 6.4–57.6 months) and AR was 87.5%. AR was significantly associated with long-term survival (odds ratio = 7, p = .01). Nine patients are currently in maintained complete response with no active treatment. Combined anti-PD-1 and SABR in oligoprogressive metastatic NSCLC or melanoma can achieve high rates of response while preventing or delaying further progression, with extended benefit in a subset of patients. This approach should be assessed in larger randomized trials including biomarker analysis to determine which patients can achieve long-term survival. Funding This line of research is funded by Fundación Científica de la Asociación Española Contra el Cáncer (“CLJUN211613ZAFR”) and Instituto de Salud Carlos III (“PI22/0816” Acción Estratégica en Salud 2022). The effect of antibiotics and proton pump inhibitors (PPIs) on the clinical outcomes of immunotherapy from our experience María José Contreras Gonzaleza,b; Lucía Navarroa,b; Javier López-Gonzáleza,b; Isidoro Barnetoa,b; Enrique Arandaa,b aClinical Management Unit of Medical Oncology, Hospital Universitario Reina Sofía, Córdoba, Spain; bMaimonides Biomedical Research Institute of Cordoba (IMIBIC). CIBERONC Recent studies suggest that loss of biodiversity in the gut microbiota, influenced by dysbacteriosis secondary to antibiotics (ATB) or proton pump Inhibitors (PPIs), may attenuate the benefit of immunotherapy. Our aim is to analyze the impact of antibiotics and PPIs on the efficacy of immunotherapy treatment in patients with non-small-cell lung cancer. We conducted a descriptive, observational, retrospective study of patients diagnosed between June 2018 and June 2020 with advanced non-small-cell lung cancer who received anti PD-1/anti PDL-1 monotherapy. The clinicopathological characteristics were collected from the clinical history. Of note was the use of PPIs and/or antibiotics during the month prior to the start of immunotherapy. Progression- free survival (PFS), defined as the median time from the start of immunotherapy to progression, was analyzed using chi-square and log rank; and overall survival (OS), from the start of immunotherapy to the last visit or death. We used SPSS v25. Of the 91 patients 41.5% received Pembrolizumab, 22.3% received Nivolumab and 36.2% received Atezolizumab. 62.6% received immunotherapy after chemoradiotherapy and 37.4% at diagnosis. 44.4% were epidermoid, 52.2% adenocarcinomas and 3.3% NOS. PD L1 expression was ≥50% in 29%, 1–50% in 28%, negative in 9% and unknown in 34%. The median age was 62 years. 80% had ECOG 0 and 71.3% had metastases in less than two sites. 74.2% received PPIs in the month prior to starting treatment and 32.6% ATB, with beta-lactams being the most commonly used (58.1%). Patients who received prior antibiotics had a median OS 14.9 months (95%CI: 8–21.9) vs. 24 (95%CI: 11.9–35.8) in the untreated group (p = .009) and a PFS 6.3 months (95%CI: 3.1–9.5) vs. 7.8 (95%CI: 4–12.6) if they did not receive ATB (p = .20). We obtained similar results in patients pre-treated with PPI. Median OS 21.3 months with PPI (95%CI:10.6–31.9) vs 27.8 (95%CI:13–38.9); (p = .045); PFS 6 (95%CI:3–8.5) vs 9 months (95%CI: 4–12.5) (p = .067). In our cohort, ATB use, and to a lesser extent PPI use, was associated with reduced efficacy of immunotherapy treatment in patients with advanced non-small cell lung carcinoma. Clinical characteristics and toxicities in patients treated with cemiplimab in a real-world setting. A center experience. Pablo Torres Mozas; Jose Antonio López López; Ana Laura Ortega Granados; Javier Requena Aguilar; Jonatan Zacarías Benoit Perejón; Juan Francisco Marín Pozo; Pedro Sánchez Rovira Hospital Universitario de Jaén. Jaén Cemiplimab is a IgG4 monoclonal antibody that binds to the programmed cell death 1 (PD-1) receptor, enhancing T-cell responses, including antitumor responses, through blocking PD-1 binding to PD-L1 and PD-L2 ligands. Its main indications are metastatic or locally advanced cutaneous squamous cell carcinoma, and advanced non-small cell lung carcinoma (aNSCLC) expressing PD-L1 in 50%. We have reviewed all patients who have received treatment with cemiplimab from February 2020 to December 2022 in our institution, a tertiary hospital in Spain. We found 18 patients, 61% men, with a mean age at diagnosis of advanced cancer of 69 years for aNSCLC patients and 75 for cutaneous tumors. 56% of patients have aNSCLC, the rest of them have head and neck. All aNSCLC patients were high PD-L1 expressors >50%, and 40% of them had PD-L1 expression >90%. The median PFS in the last data cutoff was 8 month (2–22). In 30% of the patients, the drug had to be discontinued prior to the first reassessment (after 2 month of treatment). Minor adverse events related to cemiplimab were hypertransaminemia, hematological, arthralgia all of them resolved with delays in treatment or high-dose corticosteroids. Major adverse events that require to discontinue the drug were weakness, pneumonitis and skin disorders. 16% of patients who had received at least 3 months of treatment, had to discontinue cemiplimab due to drug toxicity. After a follow-up period of at least 6 months, none of them have had disease progression despite being without treatment. Our experience with cemiplimab is still limited, especially in aNSCLC, but we are already able to detect a subgroup of patients who obtain great benefit with first line cemiplimab therapy. The toxicity profile in our patients is similar to that reported in the literature with similar rates of adverse effects grade >3, but with a higher rate of drug discontinuation. The high rate of early drug discontinuation may be due to the advanced age of the patients, especially those with cutaneous tumor. In patients with toxicity related to continued exposure to treatment, benefit may be maintained despite discontinuation of the drug. Predictive value of immune-related adverse events (irAE) in patients treated with immune checkpoint inhibitors (ICPI): observational prospective study (p-REX study) María Virginia Sánchez Becerraa; Elisabeth Jiménez Aguilarb; Estefania Zhan Zou3; Diana Moreno Muñozc; Andrea María González Lópezc; Juan Carlos Cámara Vicarioc; Susana Hernando Poloc; Alicia Hurtado Nunoc; Clara Olier Garated; Ana Cardena Gutiérrezb; Mónica Esteban Lópezb; Teresa Robles Bermejob; Verónica Sotelo Penab; Yanelis Pernas Sánchezb; Ruth Martínez Cabanesb; Araceli Rodríguez Moralesb; Elia Fernández Pérezb; Xabier Mielgo Rubiob aInstitut Gustave Roussy. Villejuif, Francia; bHospital Universitario Fundación Alcorcón. Madrid, España; cHospital Universitario Nuestra Señora de la Candelaria. Santa Cruz de Tenerife, España.; dHospital Virgen de la Luz. Cuenca, España Trials of immune checkpoint inhibitors (ICPI) in monotherapy and in combination with chemotherapy, have described two different groups of patients: the early resistance and long responders. Hence, the importance of identifying biomarkers of response. PD-L1 has been validated as a predictor of response. However, it has some limitations. Clinical, blood and histologic biomarkers have been proposed. Retrospective studies and meta-analysis have postulated that the presence of immune-related adverse events (irAEs) could have a role in predicting response. This is a prospective observational unicentric study to evaluate the predictive role of irAE in the response to ICPI. Population: patients with solid neoplasms, treated with ICPI (monotherapy/combined) in Hospital Universitario Fundacion Alcorcon, from October 2020 until July 2022. Primary endpoint was overall survival (OS) based on the presence (irAE) or absence of irAEs (non-irAE). Secondary endpoints included Progression Free Survival (PFS) and Overall Response Rate based on irAE/No-irAE. Quantitative values were expressed in frequencies, while quantitative with median/mean. Survival estimation was performed using the Kaplan-Meier method, and comparisons with Log-rank test. 48 patients were included. 45.8% received ICPI in monotherapy and 54.2% in combination. 73% anti-PD1 (16.7% nivolumab, 56.3% pembrolizumab), and 27.1% anti-PDL1 (2.1% durvalumab, 25% atezolizumab). 29.2% did not experienced irAE, 29.2% had one irAE, 20.8% two, 16.7% three and 4.2% four. 22.9% withdrew ICPI (54.5% definitive, 45.5%temporal). 50% reexperienced irAE when restarting treatment. 14.6% were hospitalized because of irAEs. There weren’t toxic deaths. 91.2% controlled disease (partial response, complete response, stable disease, pseudoprogression) in irAE and 71.4% no-irAE (p = .081). There was a favorable tendency for OS in irAE group: 12-month OS rate was of 44.8% (CI95%: 16.8%-69.7%), and 69% (CI95%: 45.1%-84.2%) for no-irAE (HR 0.476, p = .138). 12-month PFS rate was of 29% for irAE (CI95%: 6%-57.9%) and 41.2% (CI95%: 21.8%-59.7%) for no-irAE (HR 0.802, p = .581). Our conclusions are: a) there is no universal predictor of response to ICPI; b) irAE could be a biomarkers, and c) prediction of response to ICPI may come along the development of combined scores. Understanding the immune microenvironment of pancreatic ductal adenocarcinoma patients. A transcriptomic study Zoraida Andreu-Martíneza, Raquel López-Reiga, Antonio Fernández-Serraa, Isidro Machado- Puertob, María García- Floresa, María Caballero-Sotoc, Marta Trallero-Anoroc, Carmen Martínez-Lapiedrad, Fernando Martínez de Juand, Marta Ramirez-Calvo1, Tania Mazcuñan-Vitielloa, Patricia Carretero-Hinojosaa, Rafael Estevanc, Jose Antonio López-Guerreroa aLaboratory of Molecular Biology, Fundación Instituto Valenciano de Oncología, Valencia, Spain; bPathological Anatomy Department, Fundación Instituto Valenciano de Oncología, Valencia, Spain; cSurgery Service, Fundación Instituto Valenciano de Oncología, Valencia, Spain; eDigestive Service, Fundación Instituto Valenciano de Oncología, Valencia, Spain Pancreatic ductal adenocarcinoma (PDAC) prognosis and treatment response, including immunotherapy, is devastatingly poor. PDAC is an aggressive and highly heterogeneous disease that presents an immunosuppressive phenotype vaguely understood. In fact, among solid tumors, PDAC is an immunologically “cold” tumor, characterized by sparse T-cell infiltrates. Here, we studied the immune profiling of 28 Formalin-Fixed Paraffin-Embedded tissues (FFPE) of a retrospective cohort of PDAC patients by HTG EdgeSeq using a Precision ImmunoOncology panel (1392 genes). Additionally, we have performed a tissue microarray to evaluate the presence of CD4+, CD8+, PD-L1, PD-1 and CD20+ intra and peri-tumoral in the samples. The unsupervised study of immune oncogene panel results in two cluster of PDAC patients. Kaplan-Meier survival analysis indicates that cluster 1 present a poorer prognosis than cluster 2 with a median survival of 13 and 57 months, respectively, p-value = 0.0088. The differential gene expression analysis identified 478 dysregulated genes (239 up; 239 down). Then, with a more restrictive p-value (< 0.001), we selected 28 differentially expressed genes (DEGs) that also properly discriminate the two clusters of patients. Moreover, the Kaplan-Meier survival analysis in top 5 genes indicates that the over expression of DLX6 (p-value = 0.11), KRT13 (p-value = 0.037), and LAG3 (p-value = 0.014) in cluster 1 confers a worse prognosis. Finally, gene set enrichment analysis (GSEA) showed 17 pathways downregulated in cluster 1, highlighting, Leukocyte trans-endothelial migration and Toll-like receptor signaling pathways related to immune system. In conclusion, our results point to distinguish two kinds of PDAC tumors based on immune environment: “cold and hot” tumors that could be useful for the stratification of patients who may benefit from an immunotherapy strategy. Funding This work has been carried out under the framework of the ULISES project: H2020-FETOPEN-2018-2019-2020-01 Contract n°: 899708 Overcoming the tumor microenvironment acidic Ph to enhance T cell based immunotherapy Flor Navarroa; Noelia Casaresa; Celia Marín-Otala; Aritz Lasarte-Cíaa; Ángela Covo1; Marta Gorraiza; Patricia Sarrióna; Diana Llopiza; David Reparaza; Nerea Varob; Juan Roberto Rodríguez-Madoza; Felipe Prosperb; Sandra Hervás- Stubssa; Teresa Lozanoa aCentro de Investigación Médica Aplicada (CIMA) Universidad de Navarra. Pamplona. bClínica Universidad de Navarra. Pamplona The high metabolic activity of tumors leads to the acidification of the tumor microenvironment (TME) that may inhibit the antitumor T cell activity. We investigated how to make T cells more resistant to the tumor’s acid pH by modifying the function or expression of some ion exchangers and channels in order to alkalinize T cell intracellular pH in the TME, which could result in enhanced anti-tumor immune responses. We focused on acid loader chloride/bicarbonate anion exchanger 2 (Ae2), that loads acid chloride into the cytoplasm while extruding HCO3-, and Hvcn1, a selective H+ outward extruder, that alkalinizes the cytoplasm of the cell. We hypothesized that modifying the function or expression of these channels could alkalinize T cells intracellular pH in the TME for a better function First, we proposed the Ae2 inhibition function in T lymphocytes with selective chemical inhibitors like DIDs (4,4’-diisothiocyanatostilbene-2,2’-disulfonicacid). Second, we tried a genetic approach by deletion of Ae2 expression using CRISPR Cas9 technology. Also, a second strategy using Hvcn1 overexpression on T with using retrovirus transduction was performed. Finally, we designed in vivo experiments with adoptive cell transfer therapy using modified T cells (AE2 KO or Hcvn1 overexpressed) in tumor- bearing mice. First, we proposed the Ae2 inhibition function on T lymphocytes with selective chemical inhibitors like DIDs (4,4’-diisothiocyanatostilbene-2,2’-disulfonicacid). Second, we tried a genetic approach by deletion of Ae2 expression using CRISPR Cas9 technology. Also, a second strategy using Hvcn1 overexpression on T cells with retrovirus transduction was performed. Finally, we designed in vivo experiments with adoptive cell transfer therapy using modified T cells (AE2 KO or Hcvn1 overexpressed) in tumor bearing mice (Figure 1). We found that pharmacological inhibition of Ae2 with DIDS enhanced CD4+ and CD8 + T cells function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specific T cells. Notably, genetic Ae2 silencing in OVA-specific T cells improved CD4+/CD8 + T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specific T cells to overexpress Hvcn1, significantly improved T cell function in vitro, even at low pH conditions. In vivo, the adoptive transfer of OVA-specific T cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearing mice. Moreover, Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors.We found that pharmacological inhibition of Ae2 with DIDS enhanced CD4+ and CD8 + T cells function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specific T cells. Notably, genetic Ae2 silencing in OVA-specific T cells improved CD4+/CD8 + T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specific T cells to overexpress Hvcn1, significantly improved T cell function in vitro, even at low pH conditions. In vivo, the adoptive transfer of OVA-specific T cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearing mice. Moreover, Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors. Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specific lymphocytes enhances their antitumor response by making them more resistant to the acidic TME.Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specific lymphocytes enhances their antitumor response by making them more resistant to the acidic TME. Reprogramming of cancer cell metabolism by mitochondrial pyruvate carrier-1 fosters immune-mediated tumor remission Ainhoa Ruiz-Iglesiasa; Ángel García Aldeaa; Marta Gómez de Cedrónb; Rosa M. Peregila; Miguel Ángel Sánchezc; Emilia Miraa; Rosa A. Lacallea; Raquel Blancoa; Teresa Navarrod; María C. Moreno-Ortize; Ana Ramírez de Molinac; Santos Mañesb a Department of Immunology and Oncology. Centro Nacional de Biotecnología (CNB) – CSIC. Madrid. bMolecular Oncology Group. Precision Nutrition and Cancer Program. IMDEA Food Institute (CEI UAM+CSIC). Madrid. cFlow Cytometry Facility. Centro Nacional de Biotecnología (CNB) – CSIC. Madrid. dHigh-Field Magnetic Resonance Imaging and Spectroscopy Facility. Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC- UAM). Madrid. eInstituto de Investigaciones Biomédicas “Alberto Sols” (CSIC-UAM). Madrid Cancer cell metabolism is highly glycolytic. This is thought to sustain cancer cell proliferation, and to foster immunosuppression through lactate secretion to the tumor microenvironment (TME). In silico analyses indicate that downregulation of the mitochondrial pyruvate carrier (MPC) genes, the transporters of pyruvate from the cytosol to the mitochondrial matrix, is associated with poor prognosis in colon and pancreatic cancers. The aim of this thesis is the development of murine tumors overexpressing MPC1 to analyze the effects on immune populations and response in order to improve anti-tumor response alone or in combination with other therapies. Murine colon (MC38) and pancreatic (Panc02) adenocarcinoma cells were transduced with the bicistronic LeGO-iG2-MPC1-IRES-GFP lentivirus. MPC1 cell clones were selected through SeaHorse and lactate secretion analyses. Tumors were generated by s.c. inoculation of mock- and MPC1-overexpressing cells in immunocompetent and CD8 + T-cell-depleted C57BL/6, and in Rag2-Il2rγ-KO mice. CD8 + T-cell functionality in tumor-draining lymph nodes was analyzed by flow cytometry. Metabolites in the TME were determined by ex-vivo 1H HR-MAS. MPC1-overexpressing tumors showed lower incidence and growth compared to mock when implanted in immunocompetent mice. Spontaneous regression of MPC1-tumors occurred in 60% of the mice. Rechallenge of mice that rejected MPC1-tumors with MC38 mock cells resulted in no tumor implantation, indicating the induction of immune memory (Figure 1). MPC-1 overexpression boosted CD8 + T-cell-mediated immune responses compared to mock, as suggested by enhanced CD8 + T-cell activation upon restimulation of cells isolated from the tumor-draining lymph node (Figure 1). Supporting a role for the adaptive immune system, we found that: (i) the growth of MPC1-expressing tumors was recovered when implanted in CD8 + T-cell-depleted mice, and (ii) the growth of mock and MPC-1-overexpressing tumors showed no statistical differences when implanted in Rag2-Il2rγ-KO mice. 1H HR-MAS spectra analysis revealed no significant differences in lactate concentration between MPC1- and mock-tumors. Overexpression of MPC1 in cancer cells boosts a long-term anti-tumor immune response, involving the adaptive immune system. This anti-tumor effect might not be mediated by a reduction of lactate levels in the TME in MPC1-overexpressing tumors. Funding Work supported by grant 116303RB-100 (Plan Nacional 2020) and grant FPU (2019). The metabolism of diacylglycerol: a novel tool for cancer therapy Miguel Martín-Salgadoa; Juan José Sánchez-Cabezóna; Aitana Herrera-Azconaa; Rosa Liébana-Gallego; Sara Casado-Ramosa; Isabel Mérida de San Romána a Centro Nacional de Biotecnología (CNB) – CSIC. Madrid Diacylglycerol kinases (DGKs) are a family of lipidic enzymes that catalyze the conversion of diacylglycerol (DAG) into phosphatidic acid (PA). Ten isoforms have been described in mammals, of which, DGKα and DGKζ are the major isoforms expressed in T cells. DGKs are master regulators of T cell activation with which they have a fundamental role in the regulation of peripheral tolerance. Thus, pharmacological modulation of DGKs enzymatic activity is of great interest in the fight against tumors. T lymphocytes require at least three signals for proper activation: antigen recognition by the TCR, costimulatory signals and polarizing cytokines. Lack of any of them would drive T cells to anergic phenotype acquisition. Cytokines are key participants in differentiation of T cells, being common gamma chain (γc) cytokines (IL-2 and IL-15) of special relevance. IL-2 drives expansion of CD8 T cells and acquisition of cytotoxic phenotypes whereas IL-15 is mandatory for proliferation and later differentiation to memory phenotypes. While the clinical use of IL-2 has been withdrawn due to side effects, the use of IL-15 gives rise to less toxic but more limited responses in terms of effectiveness. Previous studies from our laboratory revealed a role for DGKζ in controlling IL-15 driven responses, suggesting that IL-15 (alone or in combination with TCR signals) could lead to DAG elevation. In this context, the work presented here tries to analyze the possible therapeutic advantages of IL-15 in mouse models deficient for DGKζ Total splenocytes were isolated from WT and DGKζ deficient mice and activated with plate bound anti-CD3ε, soluble anti CD28 and IL-2 or IL-15. For in vivo experiments, mice were injected with IL-15 couple to α chain. Two doses were given in two consecutive days. Three days later, bone marrow and spleen were processed for FACS analysis. Our results confirm a specific role for DGKζ in the control of cytokine driven responses. Tumor establishment assays have shown increased resistance of DGKζ deficient mice to tumor progression. Likewise, inoculation of exogenous IL-15 favors the expansion of CD8 T lymphocytes with effector features in specific niches like the bone marrow, making mobilization of great interest for tumor clearance. In conclusion, DGKs are master regulators of T cell homeostasis. Understanding the mechanisms underlaying T cell activation is essential when seeking for combinations of pharmacological agents against cancer. Here we propose a model by which DGKζ deficiency cooperates with IL-15 to promote enhance T cell activation and tumor rejection (Figure 1). Funding This work was supported by grants from the MINECO (PID2019- 108357RB-I00), the Spanish Association Against Cancer (AECC-1518), the Madrid regional government (IMMUNOTHERCAM Consortium B2017/BMD-3733) and the Aplastic Anemia and MDS International Foundation (AAMDSIF OPE01644), to IM. MMS is a predoctoral fellow of the Formation of Research Personnel (FPI) Program. BRAINTRUCK: turning NKG2D CAR-T therapy into an improved IL18 truck as treatment for pediatric central nervous system tumors Laura Clares Villaa; Lidia Pertíñeza; Alfonso Navarro Zapataa; Carmen Mestre Durána; Carla Martín Cortázara; Karima Al-Akioui Sanza; Marta Ibáñez Navarrob; Lucía Fernándezb; Jordi Minguillóna; María Puerto Moralesc; Domingo F. Barberd; Antonio Rodrígueze; Antonio Pérez Martíneza, e, f; Cristina Ferreras Puentea aInstituto para la Investigación Biomédica del Hospital Universitario La Paz (IdiPAZ). Madrid. bCentro Nacional de Investigaciones Oncológicas. Madrid. cInstituto de Ciencia de Materiales de Madrid (ICMM-CSIC). Madrid. dCentro Nacional de Biotecnología (CNB-CSIC). Madrid. eUniversidad Autónoma de Madrid. Madrid. fServicio de Hemato-Oncología Pediátrica, Hospital Universitario La Paz. Madrid. Central Nervous System (CNS) tumors are the most common solid tumors in children and the leading cause of cancer-related death in children. Although current therapies have improved their outcome, survivors present severe long-time sequelae from therapy. Chimeric Antigen Receptor (CAR) T cells arise as a therapeutic alternative in hematological malignancies but their efficacy is still a challenge in solid tumors. This is due in part to the scarce infiltration into the tumor site, the lack of therapy persistence and the immunosuppressive tumor microenvironment (TME). NKG2D CAR-T has already shown substantial benefit in osteosarcoma studies in vivo, with some limitations. This project aims to improve CAR-T cell therapy on pediatric brain tumors by generating different tools whose utility exceeds pediatric brain oncology itself. In order to overcome TME limitations, we will manipulate NKG2D CAR-T cells to release different versions of interleukin 18 (IL18) upon CAR T activation. It will transform the immunosuppressive TME, allowing immune cells to reach the tumor, proliferate and exert their cytotoxic activity. IL18 is a proinflammatory cytokine that recruits immune cells, as well as activates NK and T cells. Restriction site digestion and subsequent DNA fragment ligation has been performed in order to clone four IL18 versions under the control of the NFAT promoter and the PGK promoter. Engineered IL18 is expected to increase the protein stability and release. Cells were transfected with expression plasmids containing engineered IL18. IL18 released to the extracellular medium was quantified through ELISA. Engineered IL18 bioactivity is currently being tested through migration assays and NK activation assays. Quantifications revealed one of the IL18 modifications causes an increase in the cytokine released to the extracellular medium. Functionality comparison among the different IL18 versions is being performed. The more effective engineered IL-18 candidates will be cloned in NKG2D CAR-T. Then, we are currently developing 3D models to assess the efficacy and infiltration of the therapy. With this interdisciplinary approach we aim to improve current hurdles in CAR-T therapeutic efficacy for the treatment of pediatric brain tumors. Funding This project is founded through the Spanish Association Against Cancer (AECC) in Madrid.

