Scientific publications

Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

Sep 19, 2023 | Magazine: Frontiers in Immunology

Cristina Calviño  1 , Candela Ceballos  2 , Ana Alfonso  1   3 , Patricia Jauregui  1 , Maria E Calleja-Cervantes  4   5 , Patxi San Martin-Uriz  4 , Paula Rodriguez-Marquez  3   4 , Angel Martin-Mallo  4 , Elena Iglesias  4 , Gloria Abizanda  4 , Saray Rodriguez-Diaz  4 , Rebeca Martinez-Turrillas  3   4 , Jorge Illarramendi  2 , Maria C Viguria  2 , Margarita Redondo  2 , Jose Rifon  1   3 , Sara Villar  1 , Juan J Lasarte  6   7 , Susana Inoges  1   3   7   8 , Ascension Lopez-Diaz de Cerio  1   3   7   8 , Mikel Hernaez  3   5   7   9 , Felipe Prosper  1   3   4   7 , Juan R Rodriguez-Madoz  3   4   7


Abstract

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.

CITA DEL ARTÍCULO  Front Immunol. 2023 Sep 19:14:1270843. doi: 10.3389/fimmu.2023.1270843

Our authors

Dr. Sara Villar Fernández
Mª Erendira Calleja Cervantes
Bioinformatics Research Technician Adoptive Cellular Therapy Research Group
Dr. Patxi San Martín Uriz
Dr. Paula Rodríguez Márquez
Ángel Martín Mallo
Elena Iglesias López
Gloria Abizanda Sarasa
Saray Rodríguez Díaz
Rebeca Martínez Turrillas
Dr. Mikel
Researcher | Principal Investigator Computational Biology Program