Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia
Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We in...
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Frontiers Media S.A.
2023-09-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1270843/full |
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| author | Cristina Calviño Candela Ceballos Ana Alfonso Ana Alfonso Patricia Jauregui Maria E. Calleja-Cervantes Maria E. Calleja-Cervantes Patxi San Martin-Uriz Paula Rodriguez-Marquez Paula Rodriguez-Marquez Angel Martin-Mallo Elena Iglesias Gloria Abizanda Saray Rodriguez-Diaz Rebeca Martinez-Turrillas Rebeca Martinez-Turrillas Jorge Illarramendi Maria C. Viguria Margarita Redondo Jose Rifon Jose Rifon Sara Villar Juan J. Lasarte Juan J. Lasarte Susana Inoges Susana Inoges Susana Inoges Susana Inoges Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Mikel Hernaez Mikel Hernaez Mikel Hernaez Mikel Hernaez Felipe Prosper Felipe Prosper Felipe Prosper Felipe Prosper Juan R. Rodriguez-Madoz Juan R. Rodriguez-Madoz Juan R. Rodriguez-Madoz |
| author_facet | Cristina Calviño Candela Ceballos Ana Alfonso Ana Alfonso Patricia Jauregui Maria E. Calleja-Cervantes Maria E. Calleja-Cervantes Patxi San Martin-Uriz Paula Rodriguez-Marquez Paula Rodriguez-Marquez Angel Martin-Mallo Elena Iglesias Gloria Abizanda Saray Rodriguez-Diaz Rebeca Martinez-Turrillas Rebeca Martinez-Turrillas Jorge Illarramendi Maria C. Viguria Margarita Redondo Jose Rifon Jose Rifon Sara Villar Juan J. Lasarte Juan J. Lasarte Susana Inoges Susana Inoges Susana Inoges Susana Inoges Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Mikel Hernaez Mikel Hernaez Mikel Hernaez Mikel Hernaez Felipe Prosper Felipe Prosper Felipe Prosper Felipe Prosper Juan R. Rodriguez-Madoz Juan R. Rodriguez-Madoz Juan R. Rodriguez-Madoz |
| author_sort | Cristina Calviño |
| collection | DOAJ |
| description | Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment. |
| first_indexed | 2024-03-11T23:35:32Z |
| format | Article |
| id | doaj.art-f8de5ca01ff34ed9b089165eb0725060 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-03-11T23:35:32Z |
| publishDate | 2023-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-f8de5ca01ff34ed9b089165eb07250602023-09-20T04:42:44ZengFrontiers Media S.A.Frontiers in Immunology1664-32242023-09-011410.3389/fimmu.2023.12708431270843Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemiaCristina Calviño0Candela Ceballos1Ana Alfonso2Ana Alfonso3Patricia Jauregui4Maria E. Calleja-Cervantes5Maria E. Calleja-Cervantes6Patxi San Martin-Uriz7Paula Rodriguez-Marquez8Paula Rodriguez-Marquez9Angel Martin-Mallo10Elena Iglesias11Gloria Abizanda12Saray Rodriguez-Diaz13Rebeca Martinez-Turrillas14Rebeca Martinez-Turrillas15Jorge Illarramendi16Maria C. Viguria17Margarita Redondo18Jose Rifon19Jose Rifon20Sara Villar21Juan J. Lasarte22Juan J. Lasarte23Susana Inoges24Susana Inoges25Susana Inoges26Susana Inoges27Ascension Lopez-Diaz de Cerio28Ascension Lopez-Diaz de Cerio29Ascension Lopez-Diaz de Cerio30Ascension Lopez-Diaz de Cerio31Mikel Hernaez32Mikel Hernaez33Mikel Hernaez34Mikel Hernaez35Felipe Prosper36Felipe Prosper37Felipe Prosper38Felipe Prosper39Juan R. Rodriguez-Madoz40Juan R. Rodriguez-Madoz41Juan R. Rodriguez-Madoz42Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainHematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, SpainHematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainHematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainComputational Biology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainHematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, SpainHematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, SpainHematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, SpainHematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainHematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainImmunology and Immunotherapy Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainCancer Center Clinica Universidad de Navarra (CCUN), Pamplona, SpainHematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainCancer Center Clinica Universidad de Navarra (CCUN), Pamplona, SpainImmunology and Immunotherapy Department, Clinica Universidad de Navarra, Pamplona, SpainHematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainCancer Center Clinica Universidad de Navarra (CCUN), Pamplona, SpainImmunology and Immunotherapy Department, Clinica Universidad de Navarra, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainComputational Biology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainCancer Center Clinica Universidad de Navarra (CCUN), Pamplona, SpainData Science and Artificial Intelligence Institute (DATAI), Universidad de Navarra, Pamplona, SpainHematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainCancer Center Clinica Universidad de Navarra (CCUN), Pamplona, SpainCentro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, SpainHemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, SpainCancer Center Clinica Universidad de Navarra (CCUN), Pamplona, SpainDespite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.https://www.frontiersin.org/articles/10.3389/fimmu.2023.1270843/fullallogeneic CAR-TCRISPRtransposonAMLtranscriptomics (RNA sequencing) |
| spellingShingle | Cristina Calviño Candela Ceballos Ana Alfonso Ana Alfonso Patricia Jauregui Maria E. Calleja-Cervantes Maria E. Calleja-Cervantes Patxi San Martin-Uriz Paula Rodriguez-Marquez Paula Rodriguez-Marquez Angel Martin-Mallo Elena Iglesias Gloria Abizanda Saray Rodriguez-Diaz Rebeca Martinez-Turrillas Rebeca Martinez-Turrillas Jorge Illarramendi Maria C. Viguria Margarita Redondo Jose Rifon Jose Rifon Sara Villar Juan J. Lasarte Juan J. Lasarte Susana Inoges Susana Inoges Susana Inoges Susana Inoges Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Ascension Lopez-Diaz de Cerio Mikel Hernaez Mikel Hernaez Mikel Hernaez Mikel Hernaez Felipe Prosper Felipe Prosper Felipe Prosper Felipe Prosper Juan R. Rodriguez-Madoz Juan R. Rodriguez-Madoz Juan R. Rodriguez-Madoz Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia Frontiers in Immunology allogeneic CAR-T CRISPR transposon AML transcriptomics (RNA sequencing) |
| title | Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia |
| title_full | Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia |
| title_fullStr | Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia |
| title_full_unstemmed | Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia |
| title_short | Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia |
| title_sort | optimization of universal allogeneic car t cells combining crispr and transposon based technologies for treatment of acute myeloid leukemia |
| topic | allogeneic CAR-T CRISPR transposon AML transcriptomics (RNA sequencing) |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1270843/full |
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