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.
Introduction Efforts to improve the survival of non-Hodgkin lymphoma (NHL) patients with recurrent disease have focused primarily on the use of consolidative myeloablative autologous hematopoietic stem cell transplantation (HSCT). However, the major limitation of HSCT for NHL is the high incidence of relapse, even at maximally tolerated preparative regimen intensities. In a series of phase I studies designed to improve HSCT longterm remission rates, we have assessed the safety and feasibility of cellular immunotherapy utilizing ex vivo expanded autologous central memory (Tcm)-enrichedT cells that are genetically modified to express CD19-specific chimeric antigen receptors (CD19CAR), given in conjunction with standard of care myeloablative HSCT. Methods Here we present results from the first two studies investigating different starting cell populations and CAR constructs. The NHL1 trial utilized a starting population of CD8+ Tcm and transduced with a lentiviral vector encoding the 1st-generation CD19CAR (CD19R:zeta), consisting of a CD19-specific scFv linked to a CD3-zeta (CD19R:zeta) signaling domain. The NHL2 trial used a bulk Tcm population including both CD4+ and CD8+cells, which were transduced with lentiviral vectors encoding a 2nd-generation CD19CAR that added a CD28 costimulatory domain (CD19R:CD28:zeta) and a selectable marker for cell tracking (EGFRt). Engineered Tcm-derived CD19CAR T cells were infused 2 days after HSCT at dose levels of 25-200 x10^6 CAR T cells (dose levels in table), and all participants were followed for dose limiting toxicity (DLT) for 28 days. Both phase I studies utilized the target equivalence range design, which defines the dose escalation and de-escalation rules for determining maximum tolerated dose based on a target range of acceptable toxicity. Results NHL1 protocol (NCT01318317): Eight participants were consented and received CD8+ Tcm -derived CD19R:zeta T cell therapy. Seven patients had a diagnosis of diffuse large B cell lymphoma (DLBCL) and 1 had mantle cell lymphoma (MCL). Four of the 8 were female, and 3/8 were ≥ age 65 years. The mean age was 62 years (50-75). The median number of prior chemo/immunotherapy regimens was 3 (2-4). Two of the 8 (25%) participants had prior radiation. Five of 8 (63%) participants on NHL1 achieved a best response of CR or continuing CR. Four of 8 (50% 95% CI [16%, 84%]) participants have progressed. The progression free survival (PFS) at both 1 and 2 years is 50%, 95% CI[16%,84%] with a median follow-up of 24.7 (min=24.0, max=26.7) months. There were 2 deaths, both from disease progression. NHL2 protocol (NCT 01815749): Eight participants were consented and received Tcm-derived CD19R:CD28:zeta/EGFRt T cell therapy. Four patients had MCL, 4 had DLBCL, 3/8 were female, 2/8 were ≥ age 65 years. The mean age was 58 years (23-71). The median number of prior chemo/immunotherapy regimens was 2 (1-3). All eight NHL2 participants achieved a best response of CR or continuing CR. The PFS at 6 months is 100%, 95% CI[63%, 100%] with a median follow-up of 12.2 (min=10.0, max=14.1) months. To date 2 participants of the 8 (25%, 95% CI [3%, 65%]) have progressed (one at 6.4 months and one at 12.6 months). There was 1 death from disease progression. Both NHL1 and NHL2 trials demonstrated safety and feasibility. There were no DLTs, delayed hematopoietic reconstitution, or non-relapse mortality on either study. In NHL2, we employed bulk Tcm including both CD4+ and CD8+ cells in the CAR transduction and also added a CD28 co-stimulatory domain in the CAR design, to enhance persistence and antitumor activity. NHL2 exhibited better CAR T cell persistence compared to NHL1 T cell therapy based on area under the curve of log10copies/µg of genomic DNA from day 1 to 25 post infusion (mean difference = 14.8, 95% CI [7.4, 22.3], P<0.001) based on analysis of WPRE PCR data. Conclusions We conclude that Tcm-derived CD19CAR T cell therapy is very safe for treatment of poor-risk NHL patients undergoing autologous HSCT. We continue follow-up of these patients long-term to assess efficacy, and preliminary data are promising. Meanwhile we are exploring CAR vector design and T cell population modifications to improve the duration of anti-tumor immunity in the setting of immune reconstitution following engineered autograft. Table. Trial CAR+ Cell Dose # of Patients NHL1 25 x 10^6 1 50 x 10^6 4 100 x 10^6 3 NHL 2 50 x 10^6 3 200 x 10^6 5 Disclosures Khaled: Sequenom: Research Funding. Siddiqi:Pharmacyclics/Jannsen: Speakers Bureau; Kite pharma: Other: attended advisory board meeting; Seattle Genetics: Speakers Bureau. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Cell Medica: Membership on an entity's Board of Directors or advisory committees. Jensen***:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Forman***:Amgen: Consultancy; Mustang: Research Funding.
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