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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.

Purpose T cells engineered with chimeric antigen receptors (CARs) recognizing CD19 can induce complete remission of B cell malignancies in clinical trials; however, in some disease settings CAR therapy confers only modest clinical benefit due to attenuated persistence of CAR T cells. The purpose of the study was to enhance persistence and augment the antitumor activity of adoptively transferred CD19CAR T cells by re-stimulating CAR+ T cells through an endogenous cytomegalovirus (CMV)-specific T cell receptor. Experimental design CMV-specific T cells from CMV seropositive healthy donors were selected after stimulation with pp65 protein and transduced with clincal grade lentivirus expressing the CD19R:CD28:ζ/EGFRt CAR. The resultant bi-specific T cells, targeting CMV and CD19, were expanded via CD19 CAR-mediated signals using CD19-expressing cells. Results The bi-specific T cells proliferated vigorously after engagement with either endogenous CMVpp65 T cell receptors or engineered CD19 CARs, exhibiting specific cytolytic activity and IFNγ secretion. Upon adoptive transfer into immunodeficient mice bearing human lymphomas, the bi-specific T cells exhibited proliferative response and enhanced antitumor activity following CMVpp65 peptide vaccine administration. Conclusions We have redirected CMV-specific T cells to recognize and lyse tumor cells via CD19CARs, while maintaining their ability to proliferate in response to CMV antigen stimulation. These results illustrate the clinical applications of CMV vaccine to augment the antitumor activity of adoptively transferred CD19CAR T cells in patients with B cell malignancies.

CD62LC precursors enriched for na€ ıve and stem cell memory precursors (T N/SCM ) with that of T CM . We found that cytotoxic T cells (CTLs) derived from T CM transcribed higher levels of CD28, FOS, INFg, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of g-chain cytokines compared to CTLs derived from T N/SCM . Higher frequencies of CTLs derived from T CM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RgC null mice, CD8 C T CM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8C T CM derived CD19CAR C CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8 C T N/SCM derived counterparts. These studies support the use of T CM enriched cell products for adoptive therapy of cancer.

Multiple myeloma (MM), a plasma cell malignancy, accounts for approximately 1 percent of all cancers and slightly more than 10 percent of hematologic malignancies in the United States. Approximately 20,000 new cases will be diagnosed this year and over 11,000 people will die from this disease. Current therapies for MM often induce remission, but nearly all patients eventually relapse and die. T-cell mediated anti-tumor therapies using genetically modify T cells with specific chimeric antigen receptors (CARs) have non-overlapping activity, toxicity and tumor resistance profiles compared to conventional chemotherapeutic agents. The main challenge in designing a CAR T cell immunotherapeutic approach is identifying the best antigen for tumor targeting. CS-1 is a cell surface glycoprotein of the signaling lymphocyte activation molecule (SLAM) receptor family that is highly and selectively expressed on normal plasma cells and MM cells, with lower expression on NK cells and little or no expression on normal tissues. This unique expression pattern and proven clinical benefit of CS-1 monoclonal antibody for the treatment of relapsed MM makes CS-1 a good target for CAR T cell therapy. Central memory T cells (TCM) from PBMC were isolated using a two-step process on the AutoMACS device to first deplete CD14+, CD45RA+ and CD25+ cells, then to positively select CD62L+ cells. These TCM undergo anti-CD3/CD28 bead stimulation and transduction with a lentiviral vector encoding CS-1 CAR containing a CD28 co-stimulatory domain and two mutations on IgG4 linker CH-2 portion to ensure enhanced potency and persistence after adoptive transfer. Gene modified CS-1 CAR T cells specifically lysed MM.1S, a MM cell line, in 4-hour 51Cr release assays and all the CAR+ cells expressed 107a upon co-cultured with the MM.1S cells. To investigate the potency of the CS-1CAR T cells, 2x106 MM.1S cells that were engineered to express GFP firefly luciferase were inoculated into NSG mice by tibia injection. 7 days post tumor engraftment, 1x106 CS-1 CAR T cells were intravenously injected into the tumor bearing mice. In contrast to untreated and mock cell treated mice in which tumor progressed rapidly systemically, single intravenous infusion of CS-1 CAR T cells induced dramatic tumor regression and significantly prolonged survival. In addition to CS-1, CD44v6 and BCMA are antigens that have also been shown to be over-expressed on MM tumor cells. We therefore compared the two CARs with CS-1 CAR for their anti-MM activity. Based on our studies, targeting CS-1 resulted in the best efficacy (Figure 1) and would be an attractive strategy for development of a clinical trial. Disclosures No relevant conflicts of interest to declare.