Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy.
Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model.



Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments.



We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once adoptively transferred into CD19+ tumor-bearing mice, Akti treated CD19CAR T cells exhibited more antitumor activity than did untreated CD19CAR T cells.



Inhibition of Akt signaling during ex vivo priming and expansion gives rise to CD19CAR T cell populations that display comparatively higher antitumor activity.
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
Ryan
R
Urak
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
Miriam
M
Walter
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
Laura
L
Lim
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
ChingLam W
CW
Wong
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
Lihua E
LE
Budde
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
Sandra
S
Thomas
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
Stephen J
SJ
Forman
T cell Therapeutics Research Laboratory, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010 USA.
Xiuli
X
Wang
D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics / Q000276:immunology*; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology; D002454:Cell Differentiation / Q000276:immunology; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D005260:Female; D006801:Humans; D016219:Immunotherapy, Adoptive; D016086:Lentivirus / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000473:pathology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D051379:Mice; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology* / Q000627:therapeutic use; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology; D023041:Xenograft Model Antitumor Assays
D018941:Antigens, CD19; D018106:CD28 Antigens; D011948:Receptors, Antigen, T-Cell; D051057:Proto-Oncogene Proteins c-akt
Akt inhibitor; B cell malignancies; CAR T cell therapy