C-JUN overexpressing CAR-T cells in acute myeloid leukemia: preclinical characterization and phase I trial

Authors:
Zuo S, Li C, Sun X, Deng B, Zhang Y, Han Y, Ling Z, Xu J, Duan J, Wang Z, Yu X, Zheng Q, Xu X, Zong J, Tian Z, Shan L, Tang K, Huang H, Song Y, Niu Q, Zhou D, Feng S, Han Z, Wang G, Wu T, Pan J, Feng X
In:
Source: Nature
Publication Date: (2024)
Issue: 15(1): 6155
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Basic Research
Molecular Biology
Regenerative medicine
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofector® X-Unit
Experiment

CRISPR knockout
CRISPR/Cas9 gene editing was performed by electroporation of Cas9/ guide RNA (gRNA) ribonucleoprotein (RNP) complex using P3 Primary Cell 4D-Nucleofector™ X Kit S (Lonza). 10 µg Cas9 protein (Invitrogen) and 100 pmol chemically modified gRNA (Ubigene) in each reaction were pre-compounded for 45min at 25 °C to form RNP complexes.  Cells were suspended in RNP transfection buffer at 1.5–2 × 10^6 cells per 20 µl reaction and were electroporated in 16-well cuvette strips per the protocol of the manufacturer. After electroporation, cells were pipetted out and resuspended in a pre-warmed Mediumand expanded. The efficiency of knockdown was then evaluated by flow cytometry after 48 h. The non-targeting sgRNAs were electroporated into control cells. 

Abstract

Chimeric antigen receptor (CAR) T cells show suboptimal efficacy in acute myeloid leukemia (AML). We find that CAR T cells exposed to myeloid leukemia show impaired activation and cytolytic function, accompanied by impaired antigen receptor downstream calcium, ZAP70, ERK, and C-JUN signaling, compared to those exposed to B-cell leukemia. These defects are caused in part by the high expression of CD155 by AML. Overexpressing C-JUN, but not other antigen receptor downstream components, maximally restores anti-tumor function. C-JUN overexpression increases costimulatory molecules and cytokines through reinvigoration of ERK or transcriptional activation, independent of anti-exhaustion. We conduct an open-label, non-randomized, single-arm, phase I trial of C-JUN-overexpressing CAR-T in AML (NCT04835519) with safety and efficacy as primary and secondary endpoints, respectively. Of the four patients treated, one has grade 4 (dose-limiting toxicity) and three have grade 1-2 cytokine release syndrome. Two patients have no detectable bone marrow blasts and one patient has blast reduction after treatment. Thus, overexpressing C-JUN endows CAR-T efficacy in AML.