Mutant calreticulin enables potent and selective CAR-T cell therapy in preclinical models of myeloproliferative neoplasms

Pesini, Cecilia; Oñate, Carmen; Gil-Bellido, Mario; Sánchez Martínez, Diego; García-Martínez, Laura; Garrote, Marta; Azaceta Reinares, Gemma; Gálvez, Eva M; Pardo, Julian; Santiago, Llipsy; Olave, María Teresa; Millán, Lorena S.; Movilla Meno, Nieves; Paz Artigas, Laura; Ramirez-Labrada, Ariel; Menéndez-Jandula, Bárbara; Calvo-Pérez, Adanays; Bernal, Jorge Paúl; Garcia-Aznar, José Manuel; Roig, Francisco J; Iglesias, Eldris; Alvarez-Larrán, Alberto; Araujo-Voces, Miguel; Arias, Maykel A

doi:10.1136/jitc-2025-012706

Mutant calreticulin enables potent and selective CAR-T cell therapy in preclinical models of myeloproliferative neoplasms

Resumen: 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.
Idioma: Inglés
DOI: 10.1136/jitc-2025-012706
Año: 2026
Publicado en: Journal for immunotherapy of cancer 14, 1 (2026), e012706 [17 pp.]
ISSN: 2051-1426
Financiación: info:eu-repo/grantAgreement/ES/AEI/PID2020-113963RB-I00
Financiación: info:eu-repo/grantAgreement/ES/AEI/PID2024-157582OB-I00
Financiación: info:eu-repo/grantAgreement/ES/AEI/RYC2022-036627-I
Financiación: info:eu-repo/grantAgreement/ES/DGA/B29-23R
Financiación: info:eu-repo/grantAgreement/ES/ISCIII/B61-24
Financiación: info:eu-repo/grantAgreement/ES/DGA/LMP139-21
Financiación: info:eu-repo/grantAgreement/EC/H2020/101018587/EU/Individual and Collective Migration of the Immune Cellular System/ICoMICS
Financiación: info:eu-repo/grantAgreement/ES/ISCIII/CB21-13-00087
Financiación: info:eu-repo/grantAgreement/ES/ISCIII/PI25-00236
Financiación: info:eu-repo/grantAgreement/ES/ISCIII/RD24-0014-0015
Financiación: info:eu-repo/grantAgreement/ES/MICINN/PID2022-136554OA-I00
Tipo y forma: Article (Published version)
Área (Departamento): Area Histología (Dpto. Anatom.Histolog.Humanas)
Área (Departamento): Area Medicina (Dpto. Medicina, Psiqu. y Derm.)
Área (Departamento): Área Inmunología (Dpto. Microb.Ped.Radio.Sal.Pú.)
Área (Departamento): Área Mec.Med.Cont. y Teor.Est. (Dpto. Ingeniería Mecánica)
Área (Departamento): Área Biología Celular (Dpto. Bioq.Biolog.Mol. Celular)

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Articles > Artículos por área > Mec. de Medios Contínuos y Teor. de Estructuras
Articles > Artículos por área > biologia_celular
Articles > Artículos por área > Inmunología
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Mutant calreticulin enables potent and selective CAR-T cell therapy in preclinical models of myeloproliferative neoplasms