{
  "abstract": "Background The adoptive transfer of T cells engineered to express chimeric antigen receptors (CAR-T) has shown high efficacy and safety in treating various hematologic malignancies. However, many hematologic disorders, such as BCR::ABL1-negative myeloproliferative neoplasms (MPNs), lack effective treatment options. Some of these neoplasms are marked by a recurrent mutation that results in the expression of mutant calreticulin (mCALR), a neoantigen absent in healthy tissues, making it a highly specific and appealing target for CAR-T cell therapy.Methods Five distinct CARs were designed based on available monoclonal antibody sequences that target mCALR and were subsequently used to generate CAR-T cells. The most effective construct was selected through functional in vitro assays against mCALR-positive cell lines. Its efficacy was then evaluated in cell lines, patient-derived cells, and orthotopic xenograft models, assessing tumor burden, CAR-T cell infiltration, and animal survival. Bulk and single-cell RNA sequencing were performed on patient-derived cells and residual tumor cells from CART-treated mice, respectively, to investigate potential resistance mechanisms. The impact of the most relevant pathway alteration on CAR-T efficacy was also analyzed. Pharmacological rescue assays using targeted agents were then conducted.Results Among the five constructs, one demonstrated superior and specific cytotoxicity against mCALR-expressing cells, with no activity against mCALR-negative controls. This CAR-T cell also eliminated patient-derived MPN cells and controlled disease progression in xenograft models, which correlated with the persistence of CAR-T cells and tumor infiltration. Transcriptomic profiling of patient samples and residual tumor cells in spleens of treated mice revealed upregulation of anti-apoptotic proteins. Functional assays confirmed reduced CAR-T efficacy in Bcl-2 high cells, which was restored by co-treatment with venetoclax, indicating a viable combination approach to overcome resistance.Conclusions This study demonstrates, for the first time, the successful targeting of mCALR with CAR-T cells as a therapeutic strategy for MPNs. The chosen construct shows strong preclinical efficacy against established cell lines and patient-derived cells. Additionally, transcriptomic profiling uncovered apoptosis resistance mechanisms and supports a combination strategy with BH3 mimetics, such as venetoclax. These findings provide a compelling rationale for ongoing preclinical development and future clinical application of anti-mCALR CAR-T cells for the treatment of MPNs.",
  "authors": [
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "CIBERINFEC, Carlos III Health Institute, Madrid, Spain, Spain",
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CERTERA, Consorcio Estatal en Red para el desarrollo de Medicamentos de Terapias Avanzadas, Carlos III, Madrid, Spain"
      ],
      "name": "Cecilia Pesini"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "Department of Microbiology, Pediatry, Radiology and Public Health, University of Zaragoza, Zaragoza, Spain"
      ],
      "name": "Mario Gil-Bellido"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain"
      ],
      "name": "Lorena S Millan"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CERTERA, Consorcio Estatal en Red para el desarrollo de Medicamentos de Terapias Avanzadas, Carlos III, Madrid, Spain"
      ],
      "name": "Carmen Oñate"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "Department of Microbiology, Pediatry, Radiology and Public Health, University of Zaragoza, Zaragoza, Spain"
      ],
      "name": "Adanays Calvo-Pérez"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "CIBERINFEC, Carlos III Health Institute, Madrid, Spain, Spain",
        "Unidad de Nanotoxicología e Inmunotoxicología experimental, IIS Aragón, Zaragoza, Spain"
      ],
      "name": "Llipsy Santiago"
    },
    {
      "affiliations": [
        "Universidad San Jorge de Zaragoza, Villanueva de Gállego, Spain"
      ],
      "name": "Eldris Iglesias"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "Department of Microbiology, Pediatry, Radiology and Public Health, University of Zaragoza, Zaragoza, Spain"
      ],
      "name": "Jorge Paúl Bernal"
    },
    {
      "affiliations": [
        "Department of Microbiology, Pediatry, Radiology and Public Health, University of Zaragoza, Zaragoza, Spain"
      ],
      "name": "Miguel Araujo-Voces"
    },
    {
      "affiliations": [
        "Instituto de Investigación en Ingeniería de Aragón (I3A), Universidad de Zaragoza, Zaragoza, Spain"
      ],
      "name": "Laura Paz Artigas"
    },
    {
      "affiliations": [
        "Universidad San Jorge de Zaragoza, Villanueva de Gállego, Spain"
      ],
      "name": "Laura García-Martínez"
    },
    {
      "affiliations": [
        "Universidad San Jorge de Zaragoza, Villanueva de Gállego, Spain",
        "Biamics, Servicios de Bioinformatica SL, Zaragoza, Spain"
      ],
      "name": "Francisco J Roig"
    },
    {
      "affiliations": [
        "Instituto de Investigación en Ingeniería de Aragón (I3A), Universidad de Zaragoza, Zaragoza, Spain"
      ],
      "name": "Nieves Movilla Meno"
    },
    {
      "affiliations": [
        "Instituto de Investigación en Ingeniería de Aragón (I3A), Universidad de Zaragoza, Zaragoza, Spain",
        "Department of Mechanical Engineering, University of Zaragoza, Zaragoza, Spain"
      ],
      "name": "José Manuel Garcia-Aznar"
    },
    {
      "affiliations": [
        "Hematology Service, Miguel Servet University Hospital, Zaragoza, Spain"
      ],
      "name": "Bárbara Menéndez-Jandula"
    },
    {
      "affiliations": [
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CERTERA, Consorcio Estatal en Red para el desarrollo de Medicamentos de Terapias Avanzadas, Carlos III, Madrid, Spain",
        "Universidad de Zaragoza, Zaragoza, Spain",
        "Hematology Service, Lozano Blesa University Clinical Hospital, Zaragoza, Spain"
      ],
      "name": "María Teresa Olave"
    },
    {
      "affiliations": [
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CERTERA, Consorcio Estatal en Red para el desarrollo de Medicamentos de Terapias Avanzadas, Carlos III, Madrid, Spain",
        "Hematology Service, Lozano Blesa University Clinical Hospital, Zaragoza, Spain"
      ],
      "name": "Gemma Azaceta Reinares"
    },
    {
      "affiliations": [
        "Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain",
        "Hematology Department, Barcelona Clinic University Hospital, Barcelona, Spain",
        "Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain"
      ],
      "name": "Marta Garrote"
    },
    {
      "affiliations": [
        "Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain",
        "Hematology Department, Barcelona Clinic University Hospital, Barcelona, Spain"
      ],
      "name": "Alberto Alvarez-Larrán"
    },
    {
      "affiliations": [
        "CIBERINFEC, Carlos III Health Institute, Madrid, Spain, Spain",
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CSIC, Instituto de Carboquimica, Zaragoza, Spain"
      ],
      "name": "Eva M Gálvez"
    },
    {
      "affiliations": [
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CERTERA, Consorcio Estatal en Red para el desarrollo de Medicamentos de Terapias Avanzadas, Carlos III, Madrid, Spain",
        "Department of Microbiology, Pediatry, Radiology and Public Health, University of Zaragoza, Zaragoza, Spain",
        "ARAID, IIS Aragón, Zaragoza, Spain"
      ],
      "name": "Diego Sánchez Martínez"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "CIBERINFEC, Carlos III Health Institute, Madrid, Spain, Spain",
        "Department of Microbiology, Pediatry, Radiology and Public Health, University of Zaragoza, Zaragoza, Spain"
      ],
      "name": "Maykel A Arias"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "CIBERINFEC, Carlos III Health Institute, Madrid, Spain, Spain",
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CERTERA, Consorcio Estatal en Red para el desarrollo de Medicamentos de Terapias Avanzadas, Carlos III, Madrid, Spain",
        "Department of Microbiology, Pediatry, Radiology and Public Health, University of Zaragoza, Zaragoza, Spain"
      ],
      "name": "Julian Pardo"
    },
    {
      "affiliations": [
        "Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain",
        "CIBERINFEC, Carlos III Health Institute, Madrid, Spain, Spain",
        "Red RICOS de Terapias Avanzadas TERAV+, Carlos III Health Institute, Madrid, Spain",
        "CERTERA, Consorcio Estatal en Red para el desarrollo de Medicamentos de Terapias Avanzadas, Carlos III, Madrid, Spain"
      ],
      "name": "Ariel Ramirez-Labrada"
    }
  ],
  "title": "Mutant calreticulin enables potent and selective CAR-T cell therapy in preclinical models of myeloproliferative neoplasms",
  "uid": "2795a28f-82b0-571c-9343-e66338642a4b"
}