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.

CD19-directed chimeric antigen receptor (CD19CAR) T cell therapy has been successful in treating several B cell lineage malignancies, including B cell non-Hodgkin's lymphoma (NHL). This modality has not yet been extended to NHL manifesting in the central nervous system (CNS), primarily due to concerns for potential toxicity. CD19CAR T cells administered intravenously (IV) are detectable in cerebrospinal fluid (CSF), suggesting that CAR T cells can migrate from the periphery into the CNS, where they can potentially mediate anti-lymphoma activity. Here, we report the outcome of a subset of patients with primary CNS lymphoma (PCNSL; n=5) treated with CD19CAR T cells in our ongoing Phase 1 clinical trial (NCT02153580). All patients developed at least grade 1 cytokine release syndrome and neurotoxicity post-CAR T cell infusion; toxicities were reversible and tolerable, and there were no treatment-related deaths. At initial disease response, 3 of 5 patients (60%, 90%CI [19%,92%]) appeared to achieve complete remission, as indicated by resolution of enhancing brain lesions, and the remaining 2 patients had stable disease. Although the study cohort was small, we demonstrated that using CD19CAR T cells to treat PCNSL can be safe and feasible.

Multiple myeloma (MM) is an incurable malignancy of plasma cells even with great advances in treatment. Chimeric Antigen Receptor (CAR) directed T cell therapy, which can specifically recognize tumor associated antigens and kill tumor cells in an MHC independent manner, is a promising approach for hematological malignancy. There are several candidate antigens for CAR T cell targeting of multiple myeloma, including BCMA and CS1. Our goal is to develop novel CARs for the treatment of MM and explore the potential benefits of combinatorial therapy of CAR T cells and immunomodulatory drugs (IMiDs) such as lenalidomide. In the present study, we redirected central memory T cells to express second-generation CARs specific for either CS1 or BCMA that incorporate CD28 signaling moieties. Central memory T cells were activated by CD3/CD28 bead stimulation, transduced with lentivirus encoding the CAR construct, and expanded ex vivo. The engineered and expanded CS1 and BCMA CAR T cells exhibited similar phenotypes and comparable in vitro effector function. However, once adoptively transferred into MM tumor-bearing NOD/Scid IL2RγCnull (NSG) mice by intravenous injection of 1x10^6 CAR T cells, CS1 CAR T cells exhibited superior antitumor activity over BCMA CART cells and significantly prolonged mouse survival (P<0.01). To further improve the anti-MM activity of CAR T cell therapy, we investigated the effects of lenalidomide on CS1 CAR T cell function against MM. Central memory T cells were activated and transduced with lentivirus encoding CS1 CAR and then expanded in vitro in the presence of 0, 1 or 10mM lenalidomide for 3-4 weeks and then effector function was evaluated. We found that CD8+ CAR T cells were preferentially expanded over CD4+ CAR T cells in a dose-dependent manner. Lenalidomide-treated CAR T cells secreted higher levels of Th1 cytokines such as IFN-gamma, TNF-alpha, and IL-2, but reduced Th2 cytokines such as IL-4 and IL-10 upon antigen stimulation as compared with untreated CAR T cells. Meanwhile we observed that lenalidomide greatly improved the maintenance of T cell memory markers (CD62L, CD28, and CD27) in the culture and enhanced the formation of immune synapses between CAR T cells and MM cells. RNA-seq analysis revealed that more than 600 genes were differentially expressed among the lenalidomide treated and un-treated CD8+CAR+ T cells. Among those, expression of immune synapse related genes such as cell junction and biological assembly is significantly increased with lenalidomide treatment. Moreover, lenalidomide results in elevated gene transcrips characteristic of memory, homing and cytolytic function of CAR T cells. To test the synergistic effects, MM bearing mice were treated with a single infusion of 1x10^6 CS1 CAR T cells (i.v) on day 0 and/or 5-7.5mgkg-1 of lenalidomide daily (i.p.) initiating on day 0 for 30 days. CS1 CAR T cells and lenalidomide exhibited synergistic anti-MM activity in vivo when MM mice received combinatorial treatment. The combination therapy significantly inhibited tumor growth in vivo, prolonged mouse survival (P<0.01) and improved CAR T cell persistence in mice as compared to single-agent treatment. Taken together, these findings indicate that lenalidomide plays a co-stimulatory role in immune modulation of CAR T cells and strengthens the anti-tumor activity of CS1 CAR T cells in vivo. Rational combination of these immunotherapeutic regimens is an effective strategy and the planned clinical trial will use a combination of lenalidomide and CS1 CAR T cells for increasing treatment efficacy. Disclosures No relevant conflicts of interest to declare.

Lymphomas with central nervous system (CNS) involvement confer a worse prognosis than those without CNS involvement, and patients currently have limited treatment options. T cells genetically engineered with CD19-targeted chimeric antigen receptors (CAR) are effective against B-cell malignancies and show tremendous potential in the treatment of systemic lymphoma. We aimed to leverage this strategy toward a more effective therapy for patients with lymphoma with CNS disease. NOD-scid IL2Rgamma null (NSG) mice with CNS and/or systemic lymphoma were treated with CD19-CAR T cells via intracerebroventricular (ICV) or intravenous (IV) injection. CAR T cells isolated post-treatment were rigorously examined for phenotype, gene expression, and function. We observed that CAR T cells infused ICV, but not IV, completely and durably eradicated both CNS and systemic lymphoma. CAR T cells delivered ICV migrated efficiently to the periphery, homed to systemic tumors, and expanded in vivo, leading to complete elimination of disease and resistance to tumor re-challenge. Mechanistic studies indicated that ICV-delivered CAR T cells are conditioned by exposure to cerebrospinal fluid in the ICV environment for superior anti-lymphoma activity and memory function compared with IV-delivered CAR T cells. Further analysis suggested that manipulating cellular metabolism or pre-activating therapeutic CAR T cells with antigen ex vivo may improve the efficacy of CAR T cells in vivo. Our demonstration that ICV-delivered CD19-CAR T cells had activity against CNS and systemic lymphoma could offer a valuable new strategy for treatment of B-cell malignancies with CNS involvement.