Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy
Christopher A. Klebanoff, Joseph G. Crompton, Anthony J. Leonardi, Tori N. Yamamoto, Smita S. Chandran, Robert L. Eil, Madhusudhanan Sukumar, Suman K. Vodnala, Jinhui Hu, Yun Ji, David Clever, Mary A. Black, Devikala Gurusamy, Michael J. Kruhlak, Ping Jin, David F. Stroncek, Luca Gattinoni, Steven A. Feldman, Nicholas P. Restifo
Christopher A. Klebanoff, Joseph G. Crompton, Anthony J. Leonardi, Tori N. Yamamoto, Smita S. Chandran, Robert L. Eil, Madhusudhanan Sukumar, Suman K. Vodnala, Jinhui Hu, Yun Ji, David Clever, Mary A. Black, Devikala Gurusamy, Michael J. Kruhlak, Ping Jin, David F. Stroncek, Luca Gattinoni, Steven A. Feldman, Nicholas P. Restifo
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Research Article Immunology Oncology

Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy

Abstract

Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR–modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.

Authors

Christopher A. Klebanoff, Joseph G. Crompton, Anthony J. Leonardi, Tori N. Yamamoto, Smita S. Chandran, Robert L. Eil, Madhusudhanan Sukumar, Suman K. Vodnala, Jinhui Hu, Yun Ji, David Clever, Mary A. Black, Devikala Gurusamy, Michael J. Kruhlak, Ping Jin, David F. Stroncek, Luca Gattinoni, Steven A. Feldman, Nicholas P. Restifo

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Figure 4

AKTi limits the acquisition of a glycolytic metabolism in human peripheral blood T cells transduced with an anti-CD19 CAR.

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AKTi limits the acquisition of a glycolytic metabolism in human peripher...
(A) Two-dimensional principal component analysis (PCA) visualizing the variation in 308 metabolites detected using a metabolomic assay in anti-CD19 chimeric antigen receptor (CAR) transduced T cells expanded in AKTi or vehicle control (Veh). Percentages indicate the amount of variance captured within each principal component. Results are shown from 3 independently evaluated donors using the average of n = 5 replicates per condition and donor. (B) Ratio of lactate, an end glycolytic intermediate, detected in anti-CD19 CAR transduced T cells expanded in the continuous presence of AKTi versus Veh. The mean ratio ± SEM from n = 3 independently evaluated donors is shown with the dashed line indicating a lactate ratio of 1. (C) 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG) uptake and (D) extracellular acidification rate (ECAR) 5d after stimulation and expansion of peripheral blood T cells in the continuous presence of AKTi or Veh control. Results shown in panel C are representative of 3 independently performed experiments; results in panel D are based on cultures from n = 4 independently evaluated donors. (E) Heat map of gene expression for the 10 enzymes involved in glycolysis and the 2 primary lactate exporters, LDHA and SLC16A3, found in lymphocytes. Each column represents the average expression of a gene from 3 independently evaluated patient donors, and each row represents the indicated gene. Red and blue colors indicate relative increased and decreased expression, respectively. Data shown in panels A, B, and E are from patient-derived T cells, and panels C and D are from healthy donors (HDs). Statistical comparisons in panels C and D were performed using an unpaired 2-tailed Student’s t test. ***P < 0.001; *P < 0.05.