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A key determinant of the therapeutic potency of adoptive T cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR) redirected anti-tumor CD8+ effector T cells derived from CD45RA−CD62L+ central memory (TCM) precursors engraft long-term and reconstitute functional memory following adoptive transfer. Here, we describe a clinical-scale, closed system, immunomagnetic selection method to isolate CD8+ TCM from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS™ device to first deplete CD14+, CD45RA+ and CD4+ cells from PBMC, and then to positively select CD62L+ cells. The average purity and yield of CD8+CD45RA−CD62L+ TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8+ TCM are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in IL-2/IL-15 over 3–6 weeks. The resulting CD8+ TCM-derived effectors (TE(CM)) are polyclonal, retain expression of CD62L and CD28, exhibit CAR redirected anti-tumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NSG mice. Adoptive atherapy using purified TCM cells are now the subject of an FDA authorized clinical trial for the treatment of CD19+ B-cell malignancies, and three clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.

Multiple myeloma remains an incurable malignancy of plasma cells despite considerable advances in treatment. The purpose of the study was to develop novel chimeric antigen receptors (CAR) for the treatment of multiple myeloma and explore combinatorial therapy using CAR T cells and immunomodulatory drugs such as lenalidomide for increasing treatment efficacy. We redirected central memory T cells to express second-generation CAR-specific for CS1 and adoptively transferred them into multiple myeloma tumor-bearing mice to test their anti-multiple myeloma activity. CS1 CAR T cells were transduced and expanded in the presence of lenalidomide The phenotype and effector function of CS1 CAR T cells treated with and without lenalidomide were compared. Finally, CS1 CAR T cells and lenalidomide were administered to treat multiple myeloma-bearing mice as combinatorial therapy. CS1 CAR T cells exhibited efficient antitumor activity when adoptively transferred into mice. Mechanistic studies indicated that the addition of lenalidomide during CS1 CAR T-cell expansion enhanced the immune functions of CS1 CAR T cells, including cytotoxicity, memory maintenance, Th1 cytokine production, and immune synapse formation. Furthermore, lenalidomide enhanced the antitumor activity and persistence of adoptively transferred CS1 CAR T cells The study demonstrates that lenalidomide improves the anti-multiple myeloma properties of CS1-directed CAR T cells and provides a basis for a planned clinical trial using the combination of lenalidomide with engineered T cells against CS1 in relapsed myeloma. .

BackgroundInsufficient 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.MethodsVarious 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.ResultsWe 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.ConclusionsInhibition of Akt signaling during ex vivo priming and expansion gives rise to CD19CAR T cell populations that display comparatively higher antitumor activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0227-4) contains supplementary material, which is available to authorized users.