Activity Of Chimeric Antigen Receptor Research Articles

“Switchable chimeric antigen receptor T cells: a novel universal chimeric antigen receptor platform for a safe control of T-cell activation”

In a recent issue of PNAS, Rodgers et al. present an elegant study describing the development of a bifunctional antibody switch able to control retargeting and activation of chimeric antigen receptor (CAR)—engineered T cells, offering a potential additional safety tool compared to standard CAR platforms (1). Indeed, despite the recent striking responses obtained by adoptive transfer of CAR-T cells in relapsed and refractory patients affected by CD19+ malignancies, difficulties have emerged in the ability of controlling both CAR T-cell pharmacokinetic and pharmacodynamic properties (2-6).

Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level.

BackgroundThe poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC.MethodsThe aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo.ResultsWe found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα.ConclusionsTaken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0285-y) contains supplementary material, which is available to authorized users.

Abstract 2296: Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models

Abstract Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells for treatment requires ex vivo culture to facilitate CAR gene transfer and to achieve a therapeutic dose of the modified cells. Recent data suggests that specific T cell subtypes can provide enhanced anti-tumor efficacy, spurring efforts to optimize the production of therapeutic T cells via the cumbersome physical isolation of central memory T cells or culture in cytokines such as IL-7 and IL-15. Here we explored the potential for a simple culture modification to improve the therapeutic potential of CAR T cells without adding manufacturing complexity. To this end, we produced CAR T cells specific to B cell maturation antigen (BCMA) using standard IL-2 culture conditions supplemented with a PI3K inhibitor, or with IL-7 and IL-15 in place of IL-2. The in vivo activity of CAR T cells was studied in mouse models of human Burkitt's lymphoma (Daudi) and multiple myeloma (RPMI-8226), both of which express BCMA. In the Daudi model, NSG mice were injected intravenously with 2 × 106 tumor cells and allowed to accumulate a large tumor burden to model late stage disease observed in relapsed and refractory lymphoma. In this advanced disease model, anti-BCMA CAR T cells (4 × 106/mouse) cultured either in IL-2 or IL-7 and IL-15 had little or no effect on tumor growth (p = 0.22 and 0.23, respectively) and all mice succumbed to tumors within two weeks of treatment. In contrast, all animals treated with the same number of anti-BCMA CAR T cells cultured with PI3K inhibition survived and had complete long-term tumor regression (p = 0.003). The same anti-BCMA CAR T cells were studied in a model of multiple myeloma. NSG mice were injected subcutaneously with 107 RPMI-8226 cells and 22 days later received a single administration of anti-BCMA CAR T cells (4 × 105/mouse) cultured under various conditions. In this model, tumor regression occurred regardless of in vitro culture conditions. To model tumor relapse and evaluate CAR T cell durability, surviving animals were re-challenged with RPMI-8226 cells on the opposite flank two weeks after initial tumor clearance. In contrast to other conditions, all animals treated with anti-BCMA CAR T cells cultured with PI3K inhibition were protected against subsequent tumor challenge (p = 0.005). This improved therapeutic activity of anti-BCMA CAR T cells cultured with PI3K inhibition was associated with an increased frequency of CD62L+ CD8+ T cells in the drug product (p < 0.001) suggesting enrichment of this distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate an improved anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Citation Format: Shannon Grande, Molly R. Perkins, Amanda Hamel, Holly M. Horton, Fay Eng, Claire J. Rhodes, Tracy E. Garrett, Sara M. Miller, John W. Evans, Howard J. Latimer, Christopher Horvath, Michael Kuczewski, Kevin Friedman, Richard A. Morgan. Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2296.

Preserved Activity of CD20-Specific Chimeric Antigen Receptor–Expressing T Cells in the Presence of Rituximab

CD20 is an attractive immunotherapy target for B-cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation is that residual serum rituximab might block CAR binding to CD20 and thereby impede T cell-mediated anti-lymphoma responses. The activity of CD20 CAR-modified T cells in the presence of various concentrations of rituximab was tested in vitro and in vivo CAR-binding sites on CD20(+) tumor cells were blocked by rituximab in a dose-dependent fashion, although at 37°C blockade was incomplete at concentrations up to 200 μg/mL. T cells with CD20 CARs also exhibited modest dose-dependent reductions in cytokine secretion and cytotoxicity, but not proliferation, against lymphoma cell lines. At rituximab concentrations of 100 μg/mL, CAR T cells retained ≥50% of baseline activity against targets with high CD20 expression, but were more strongly inhibited when target cells expressed low CD20. In a murine xenograft model using a rituximab-refractory lymphoma cell line, rituximab did not impair CAR T-cell activity, and tumors were eradicated in >85% of mice. Clinical residual rituximab serum concentrations were measured in 103 lymphoma patients after rituximab therapy, with the median level found to be only 38 μg/mL (interquartile range, 19-72 μg/mL). Thus, despite modest functional impairment in vitro, the in vivo activity of CD20-targeted CAR T cells remains intact at clinically relevant levels of rituximab, making use of these T cells clinically feasible. Cancer Immunol Res; 4(6); 509-19. ©2016 AACR

198. CD4+ IL13Rα2-Specific CAR T Cells Exhibit Potent Effector Function Against Glioblastoma

Adoptive T cell transfer, including chimeric antigen receptor (CAR) T cell therapy, has been successfully applied to the treatment of multiple types of cancer, but the variation in response rates among patients remains an important issue. Recent studies have demonstrated that CAR T cells vary in function and persistence, depending on the specific T cell subpopulation that is engineered and the phenotype of the final T cell product. Specifically, a starting population of central memory T cells (Tcm) has been shown to improve persistence and efficacy of CAR T cell therapy. Tcm consist of both CD4+ and CD8+ pools, but little is known about the contribution of each subset for CAR T cell function. Notably, although CD8+ T cells have long been considered the primary cytotoxic effector population, CD4+ T cells have also been shown to mediate anti-tumor response against some solid tumors which is CD8-independent. It is thus critical to identify the optimal composition of CD4+ and CD8+ CAR T cells for mediating the maximal anti-tumor effect. Here, using both in vitro and in vivo glioblastoma (GBM) models, we examined the anti-tumor activity of IL13Rα2-specific CAR T cells engineered from purified CD4+ or CD8+ Tcm pools. Surprisingly, we discovered a superior antitumor response from CD4+ IL13Rα2-CAR T cells as compared to CD8+ CAR T cells. After co-culture with primary patient-derived GBM cells in vitro, CD4+ CAR T cells displayed more potent tumor killing as compared to CD8+ CAR T cells, especially when effector cell numbers were more limited. Consistently, CD4+ CAR T cells maintained better effector function in vitro when re-challenged with tumor. These CD4+ CAR T cells produced higher levels of cytokine as compared to CD8+ T cells, a difference that was further maintained after tumor re-challenge. We also assessed whether the CD4 versus CD8 composition of the IL13Rα2-CAR T cell product would impact therapeutic efficacy in vivo using primary patient-derived GBM lines orthotopically implanted in NSG mice. When injected intracranial into GBM-bearing mice, the CD4+ CAR T cells resulted in durable antitumor efficacy, mediating tumor-free survival for over 200 days in 7of 7 experimental animals, which was unaffected by mixing with 10% of CD8+ CAR T cells. While CD8+ CAR T cells, as well as 50%-50% or 10%-90% CD4-CD8 mixed populations, also mediated significant antitumor activity, the majority of mice recurred and showed a less durable therapeutic response. These results demonstrate that CD4+ CAR T cells can have potent effector function and suggest that, for at least IL13Rα2-CARs, CD4+ T cells mediated superior anti-tumor immune responses as compared to CD8+ T cells. These studies provide important insight to further elucidate the optimal composition of the CAR T cell product for the treatment of cancer.

670. A Novel Approach to Expand Antigen Recognition in Chimeric Antigen Receptors

Chimeric antigen receptor (CAR) technology, although very promising, is limited in its application by the availability of target antigens. We propose to expand the pool of potentially targetable cell-surface antigens using the variable lymphocyte receptor (VLR) of the sea lamprey as the antigen recognition region of the CAR. VLRs represent the functional region of the lamprey and hagfish adaptive immune system. They are single chained and variable length, crescent shaped proteins that are produced by assembly of leucine-rich repeat cassettes to form a gene encoding region capable of exceeding 1015 unique variations. Due to their difference in structure compared to Ig based antibodies, VLRs bind antigen in a geometrically dissimilar manner. This unique property of VLRs allows them the ability to bind antigen epitopes that may not typically be bound by scFvs, the result being a potentially expanded repertoire of tumor cell target antigens that may be used in CAR design and application. In our studies, VLRs have been successfully developed from immunized lampreys and target cell specific VLRs have been cloned for several different antigens including VLRs specific for cancer cell lines and purified proteins. This is accomplished using yeast surface display combined with flow sorting and results in monoclonal VLRs specific for the cell line with which the lampreys were immunized. The functionality of the VLR was initially demonstrated in Jurkat cells transduced with a previously generated VLR specific for the B-cell receptor of the mouse tumor line, BCL. CAR protein expression from whole cell lysates of transduced Jurkat cells showed expression of CAR protein. CAR cell surface expression was also confirmed in Jurkat cells using a construct co-expressing GFP preceding a P2A sequence with >90% cells GFP positive. Transduced CAR-Jurkat cells showed upwards of 85% activation in co-culture assays with target cells. In transduced but not co-cultured cells, activation was <5% and activation in naive cells co-cultured with BCL cells was <2%. These results establish the ability of these cells to effectively produce and express VLR-CAR protein. The BCL VLR-CAR construct was also shown to be functional in several different types of cytotoxic effector cells including gamma delta T-cells and NK-92 cells, where target cell killing was increased significantly over non-transduced cells, indicating that target cell specific toxicity is mediated through the VLR-CAR. From these results, showing that VLRs function effectively as the antigen recognition region of the CAR construct we have concluded that VLRs can serve as a unique alternative for directing CAR activity toward specific effector cells.

Abstract IA07: Chimeric antigen receptor T cells for cancer immunotherapy

Abstract Background: T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR structure consists of two fundamental domains – the antigen binding portion (most commonly single chain variable fragment (scFv) derived from a monoclonal antibody) joined to one or more intracellular T cell signaling domains. To be effective CAR T cells must expand, persist, exhibit enduring anti-tumor cytotoxicity, withstand and/or counteract an immunosuppressive tumor microenvironment, and overcome targeted tumor antigen escape following infusion. T cells targeting the CD19 antigen is a novel therapeutic approach for patients with hematologic malignancies. To this end, we have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We now extend this novel treatment approach by testing of our CD19-specific CAR in a multicenter trial for children and young adults with relapsed CD19+ B-ALL. Patients and Methods: Pediatric and young-adult patients with very high risk (VHR) or R/R CD19+ B-ALL were eligible for enrollment and leukapheresis. Patients with R/R B-ALL were eligible for infusion if they demonstrated residual disease following salvage chemotherapy. Once eligibility was determined isolation and transduction of T cells was performed using a retroviral vector encoding a CD19-specific CAR comprised of a CD19-specific scFv and the CD28 and CD3ζ signaling domains (termed 19-28z). All patients received conditioning chemotherapy followed 2 days later by 1x106 3x106 19-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells. Post-treatment response was assessed at day 14-28 bone marrow aspirate including multiparameter flow cytometry for minimal residual disease (MRD). Results: To date, 28 patients with have been enrolled on protocol with a median age of 11.5 years (range 9 months to 20 years) at time of T cell collection. We have treated 11 patients with 19-28z CAR T cells with a median age 14.9 years (range 3-22 years). Patients received a dose of 1-3 x 10^6 CAR T cells/kg and complete response (complete remission or complete remission with incomplete count recovery) occurred in 7/11 (64%) patients. Significantly, correlations with response included lower disease burden (as assessed by bone marrow cellularity; p&amp;lt;0.004) and fold expansion following CD3/28 bead activation during the generation of CAR T cells (p&amp;lt;0.03). Specifically, BM cellularity ≤50% and fold expansion (average &amp;gt;135 vs &amp;lt;40 fold expansion) was seen in responders compared to non-responders. Pre-collection peripheral blood absolute lymphocyte count, lymphocyte proliferative response (phytohemagglutinin proliferation assay), and CD3 absolute count did not correlate with response in this small sample size. Development of fever and cytokine release syndrome (CRS) occurred in responders including grade I-II (n=3) and grade III-IV (n=4). Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of CRS. Elevated serum cytokines (notably IFN-γ and IL-6) was noted in patients with CRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T cell detection within 1-2 weeks following infusion with gradual contracture over 1-2 months. Conclusions: These early results demonstrate the feasibility and significant clinical impact of CAR T cells in patients with R/R B-ALL. In an effort to more rapidly generate statistically relevant data, demonstrate the exportability of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL we have expanded this trial to include a collaborating institution. The objective of our trial is not to provide an intent-to-treat cohort, but rather demonstrate the tolerability of this technology in patients with relapsed B-ALL. Furthermore, patients meeting disease eligibility were not pre-screened for lymphocyte function prior to collection and/or treatment. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. Citation Format: Kevin J. Curran, Lewis B. Silverman, Rachel Kobos, Nancy A. Kernan, Steven P. Margossian, Jae H. Park, Craig S. Sauter, Victoria Szenes, Xiuyan Wang, Richard J. O'Reilly, Michel Sadelain, Isabelle Riviere, Reiner J. Brentjens. Chimeric antigen receptor T cells for cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA07.

Preservation of CD20-Specific Chimeric Antigen Receptor T Cell Function in the Presence of Residual Rituximab

BACKGROUNDThe CD20 antigen is an attractive immunotherapy target for B cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation of this approach is that residual serum rituximab from prior chemoimmunotherapy regimens might block CAR binding to CD20 and prevent T cell mediated anti-lymphoma responses. However, previous data from our group and others have suggested that CD20 CAR+ T cell function is only partially blocked by anti-CD20 antibody (Ab), and T cell function in the setting of anti-CD3 x anti-CD20 bispecific Ab is not blocked by rituximab levels of up to 100 μg/ml. We have further tested the impact of various concentrations of rituximab on CD20-CAR T cell activity in vitro and in vivo. METHODSCD3+ T cells (proliferation and cytokine assays) or CD8+ selected T cells (cytotoxicity assays) were isolated from healthy donors, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 2nd or 3rd generation anti-CD20 CAR constructs (Leu16-28-ζ, Leu16-28-BB-ζ, or fully human 1-5-3-NQ-28-BB-ζ). Functional assays, performed using target cells pre-incubated for 30 min. with varying concentrations of rituximab, included a CFSE assay to assess CAR T cell proliferation, Luminex assays for cytokine secretion, and a 5-hour standard 51 chromium release assay for cytotoxicity. Target cells included K562 cells transduced to express CD80 with or without CD20 (denoted "K80" and "K80-20"), Raji, Daudi, Granta, Rec-1, and FL-18 lymphoma cells. K80-20 cell lines expressing low, medium, and high CD20 were established by limiting dilution cloning.For in vivo experiments, NOD/SCID/γ-/- (NSG) mice were inoculated i.v. with rituximab-resistant Raji-ffLuc lymphoma cells. After 5 days, rituximab was administered i.p. at 25 μg/ml or 200 μg/ml, and then at 24 hours after rituximab administration CAR+ central memory T cells expressing the 1-5-3-NQ-28-BB-ζ vector were injected i.v. Tumor growth was measured with bioluminescence imaging twice weekly and mice were followed for survival. RESULTSThe availability of CD20 binding sites on Ramos lymphoma cells pre-incubated with various concentrations of rituximab was assessed with flow cytometry, and as expected, we found a dose-dependent blockade of CD20, with complete blockade at 50 μg/ml rituximab at 4°C. However, when anti-CD20-PE was incubated at 37°C, low-level CD20 binding could occur even at 200 μg/ml of rituximab. Despite the low number of available CD20 binding sites after rituximab, proliferation of CFSE-labeled CAR+ T cells was largely unimpaired in rituximab concentrations up to 200 μg/ml. In contrast, cytokine secretion was impaired in a dose-dependent manner, although even at 100 μg/ml of rituximab, interferon-γ, interleukin-2, and tumor necrosis factor a were still produced at 34-51%, 70-92%, and 79-108% of baseline levels, respectively. Cytotoxicity also decreased with increasing rituximab concentration but >75% of baseline cytolytic activity was retained at 100 μg/ml. We hypothesized that the level of CD20 expression on target cell lines might impact sensitivity to rituximab blockade. Using K80-20 cells with low, medium, or high CD20 expression we found that cytokine secretion and cytotoxicity (but not proliferation) were highly impaired upon stimulation with CD20low target cells, whereas T cell function remained completely intact when CD20high cells were used as targets.In vivo, mice bearing rituximab-refractory Raji-ffLuc tumors experienced only slight delay of tumor growth when treated with either low or high doses of rituximab, and mice treated with T cells alone had significant clearance of tumor. In mice that received low or high-dose rituximab prior to T cell infusions, tumor rejection and survival prolongation were equivalent to or better than that observed with mice receiving T cells alone (see figure). CONCLUSIONWe have shown that the in vitro and in vivo activity of CD20-targeted CAR T cells is minimally impacted after rituximab, despite a low number of available CD20 binding sites. These data suggest that residual serum rituximab levels will not present a significant impediment to CD20-targeted CAR therapy in patients who have received rituximab-containing chemotherapy regimens. [Display omitted] DisclosuresJensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Gopal:Merck: Research Funding; BioMarin: Research Funding; Seattle Genetics: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Piramal: Research Funding; Millenium: Honoraria, Research Funding; BMS: Research Funding; Janssen: Consultancy; Emergent/Abbott: Research Funding; Sanofi-Aventis: Honoraria. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Till:Pfizer: Research Funding; Roche/Genentech: Research Funding.

Implications of Minimal Residual Disease Negative Complete Remission (MRD-CR) and Allogeneic Stem Cell Transplant on Safety and Clinical Outcome of CD19-Targeted 19-28z CAR Modified T Cells in Adult Patients with Relapsed, Refractory B-Cell ALL

Background: We have previously reported high anti-tumor activity of autologous T cells genetically modified to express 19-28z chimeric antigen receptor (CAR) targeting CD19 in adult patients with relapsed or refractory (R/R) ALL (Park et al. ASH 2014). Herein, we further report the long-term outcome of a larger cohort from our phase 1 clinical trial in adults with R/R ALL (NCT01044069) with a focused analysis on the role of post-treatment minimal residual disease (MRD) negativity as a predictive marker of survival as well as the effect of allogeneic hematopoietic stem cell transplant (allo-HSCT) prior to or after CAR T cell infusion on safety and clinical outcome. Patients and Methods: Adult patients with R/R B-cell ALL (B-ALL) were enrolled. Eligible patients underwent leukapheresis, and T cells were transduced with a retroviral vector encoding a CAR comprising a CD19-specific scFv and CD28 and CD3ζ signaling domains (19-28z). All patients received lymphodepleting chemotherapy followed 2 days later by 1x106 - 3x106 19-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells. Post-treatment MRD was assessed at day 14-28 by multiparameter flow cytometry in bone marrow (BM) samples. Results: 44 patients have been treated to date. The median age was 45 years (range, 22-74). 14 patients (32%) had Philadelphia chromosome positive (Ph+) ALL (T315I mutation in 5 patients), 17 patients (39%) had prior allo-HSCT, and 24 patients (55%) had ≥ 3 prior lines of ALL therapy. Of the 44 patients, 43 patients were evaluable for response. At the time of 19-28z CAR T cell infusion, 22 of the 43 patients (51%) had morphologic disease (≥5% blasts in BM or measurable extramedullary disease) and the remaining 21 patients had minimal disease (&lt;5% blasts in BM). 36 patients (84%) were in complete remission (CR) after 19-28z CAR T-cell infusion. MRD analysis was performed in 35 of 36 CR patients, and 29 of these 35 patients (83%) achieved an MRD-negative CR (MRD-CR). As of July 13, 2015, the median follow-up was 4.2 months (range 1-45), with 16 patients having at least 6 months of follow-up. Responses appear durable with 7 patients remaining disease-free beyond 1 year up to 45 months. A median overall survival (OS) of all patients and patients who achieved MRD-CR is 8.5 months and 10.8 months, respectively. Post-treatment MRD status emerged as a strong predictive marker of OS: OS at 6 months was 76% (95% CI: 51-89) in the MRD-CR cohort vs. 14% (95% CI: 8-45) in the MRD+CR cohort. In contrast, allo-HSCT after achieving CR with CAR T cell infusion did not affect the survival rate. Of the 36 patients in CR following the T cell infusion, 12 patients underwent allo-HSCT. OS at 6 months was 70% (95% CI: 33-89) in patients who underwent post-CAR allo-HSCT vs. 64% (95% CI: 36-82) in patients who did not get allo-HSCT after CAR T cells. Comparing baseline disease characteristics of patients who had prior allo-HSCT before the CAR T cell treatment vs. no prior allo-HSCT, patients who had prior allo-HSCT (n=17) were similar in age (median age 45 vs. 46), but had higher disease burden (65% with morphologic disease vs. 44%), were more heavily pretreated (59% of patients with ≥4 lines of therapy vs. 15%), and included more high-risk disease (41% with Ph+ ALL vs. 26%). However, there was no statistically significant difference in CR rates (75%, CI: 48-93 vs. 89%, CI: 71-98), incidences of severe cytokine release syndrome (24% vs. 22%), and OS at 6 months (57% vs. 60%) between these two cohorts. Fewer patients who had prior allo-HSCT underwent another allo-HSCT following CAR T cell infusion: 2 patients vs. 10 patients with no prior all-HSCT. Although no obvious case of graft-versus-host disease (GvHD) was noted, one patient experienced a grade 3 gastrointestinal toxicity that may have been related to GvHD. Conclusions: These data confirm the potent anti-tumor efficacy of 19-28z CAR T cells (JCAR015) in adult patients with R/R ALL. MRD negativity following the 19-28z CAR T cell treatment is highly predictive of survival, and allo-HSCT post-CAR T cell infusion had no significant impact on survival. Furthermore, 19-28z CAR T cells appear to be safe in patients who had prior allo-HSCT, and may represent an attractive alternative option to second allo-HSCT. These findings are being confirmed in an ongoing multi-center, pivotal phase 2 trial evaluating JCAR015 in adult patients with R/R ALL. Disclosures Park: Amgen: Consultancy; Genentech: Research Funding; Juno Therapeutics: Other: Advisory Board, Research Funding. Riviere:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Curran:Juno Therapeutics: Consultancy. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Other: Co-Founder, stockholder, Patents &amp; Royalties: Licensed patents on CARs. Brentjens:Juno Therapeutics: Other: Co-founder, stockholder and consultant.

Bypassing the Constraint for Chimeric Antigen Receptor (CAR) Development in T-Cells Expressing the Targeted Antigen: Improvement of Anti-CS1 CAR Activity in Allogenic TCRa/CS1 Double Knockout T-Cells for the Treatment of Multiple Myeloma (MM)

Adoptive immunotherapy with autologous T-cells expressing chimeric antigen receptors (CARs) targeting CD19 has achieved long-term remissions in patients with B cell leukemia, pointing out that CAR technology may become a new alternative in cancer treatment. In this work we assessed the feasibility of targeting the CS1 antigen (SLAMF7) for the treatment of Multiple Myeloma (MM).MM is a B-cell neoplasia characterized by clonal expansion of malignant plasma cells in the bone marrow. Even if currently available therapies can improve overall survival, MM still remains incurable in most patients. Immunotherapy against MM is therefore an area in which extensive research is being made, with novel antigenic targets being considered. Among these is the CS1 glycoprotein, which is highly expressed on tumor cells from most patients with MM. However, CS1 is also expressed on normal CD8+ T-cells, which may be problematic for a CAR-based approach as antigen-expressing T cells will be targeted, impacting both the number and the phenotype of the final CAR T cell population.To circumvent this issue we have used our highly-efficient transcription activator-like effector nuclease (TALEN) gene-editing technology to inactivate CS1 in T-cells prior to transduction with a viral vector encoding an anti-CS1 CAR. Our results demonstrate that while non-gene-edited T-cells expressing an anti-CS1 CAR display limited cytolytic activity against MM cell lines, and resulted in a progressive loss of CD8+ T-cells. CS1-gene-edited CAR cells display significantly increased cytotoxic activity, with the percentage of CD8+ T-cells remaining unaffected. In addition, experiments in an orthotopic MM mouse model showed that CS1 disrupted T-cells were able to mediate an in vivo anti-tumoral activity.Subsequently, we have utilized this strategy for CS1 in the context of our allogeneic "off-the-shelf" engineered CAR+ T-cell platform. This allogenic platform utilizes TALEN gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating their potential to mediate Graft versus Host Disease (GvHD). We have previously shown that editing of the TRAC gene can be achieved at high frequencies, allowing efficient production of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. Our results also show that multiplex genome editing is possible and can lead to the production of double KO (TRAC and CS1) T-cells, allowing large scale manufacturing of allogeneic, non alloreactive CS1 specific T-cells with enhanced antitumor activity. Moreover, these allogenic T-cells could be easily available for administration to a large number of MM patients. DisclosuresGaletto:Cellectis SA: Employment. Chion-Sotinel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

BackgroundNY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC).Methodology/Principal FindingsWe demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157–165) peptide specific chimeric antigen receptor (CAR) CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels.Conclusions/SignificanceThese results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

Eradication of B-ALL using chimeric antigen receptor–expressing T cells targeting the TSLPR oncoprotein

AbstractAdoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell–associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.

Safety and antitumor activity of chimeric antigen receptor modified T cells in patients with chemotherapy refractory metastatic pancreatic cancer.

3007 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive microenvironment with a scarcity of effector T cells. Adoptive T cell therapy to restore anti-tumor...

Chimeric antigen receptors based on low affinity mutants of FcεRI re-direct T cell specificity to cells expressing membrane IgE (HYP5P.321)

Abstract IgE is the key mediator of allergic immune responses. Targeting the source of IgE, the IgE-expressing B cells, represents an attractive therapeutic approach for atopic diseases. An adoptive T cell therapy (ACT) approach that redirects T cell killing to IgE-expressing B cells is becoming increasingly feasible due to the recent advance of ACT in efficacy and safety. Here, we designed chimeric antigen receptors (CARs) based on FcεRIα and tested their abilities to recognize cells expressing the transmembrane form of IgE (mIgE), a marker for IgE-expressing B cells. The ectodomains of the wild type (WT) and six low affinity mutants of FcεRIα were fused with CD3ζ to form WT and mutant CARs. When expressed on Jurkat cells, four of the six mutant CARs mediated robust T cell responses to U266 myeloma cells expressing mIgE, and the WT CAR mediated lower T cell responses. The presence of free IgE significantly inhibited the activity of WT CAR but did not affect mutant CARs. CAR+ Jurkat cells did not respond to cells with IgE bound through FcεRI or FcεRII (CD23). CAR+ Jurkat cells did not lead to degranulation of LAD2 mast cells with IgE bound through FcεRI. Taken together, CARs based on low affinity FcεRIα mediated robust and specific T cell responses to mIgE+ cells, even in the presence of free IgE, but not to cells with IgE bound through FcεRII or FcεRI. These CARs therefore have the potency and specificity required for targeting IgE-expressing B cells in ACT for atopic diseases.

21. Non-CD3-Based Chimeric Antigen Receptor (CARs) with Enhanced Anti-Tumor Activity in Solid Tumors

Chimeric antigen receptors (CARs) based upon a single chimeric molecule bearing an antigen binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors CD28 or 4-1BB provide a potent method for engineering T cell cytotoxicity towards tumors. We describe a simple chimeric receptor based upon a killer immunoglobulin-like receptors (KIRs) normally expressed by natural killer (NK) cells and T cells. Constructed by fusing a single chain variable fragment (scFv) to the transmembrane and cytoplasmic domain of a KIR, we show that a KIR-based CAR targeting multiple tumor-related antigens triggers antigen-specific cytotoxic activity and cytokine production that is comparable to CD3ζ-based CARs with antigen-induced proliferation that is independent of additional costimulation. Using a solid tumor xenograft model that is highly resistant to CD3ζ-based CAR-targeted T cells, we further demonstrate that KIR-based CAR-targeted T cells can induce tumor regression despite in vivo persistence of CD3ζ-based CAR-modified T cells with CD28 and 4-1BB costimulatory domains. We show that the enhanced activity of the KIR-based CAR is associated with less tumor-induced T cell hypofunction that has previously been demonstrated with CD3ζ-based CARs. These data support the future clinical evaluation of a KIR-based CARs especially within solid tumors.

517. Improving CAR T Cell Efficacy for Solid Tumors By Nanogel-Based Delivery of Immunomodulatory Proteins

Adoptive T cell therapies utilizing T lymphocytes transduced to stably express an anti-CD19 chimeric antigen receptor (CAR) have shown remarkable success against refractory B cell malignancies in multiple clinical trials. Studies are underway to adapt this technology towards the treatment of solid tumors, however, the immunosuppressive tumor microenvironment of many solid tumors may represent a significant barrier to CAR T cell efficacy. In this study, we investigate a means to enhance CAR T cell function by loading them with nanogel “backpacks” containing the T and NK cell stimulatory molecule IL-15N72D:IL-15RaSu/Fc superagonist complex (IL-15sa, also referred to as ALT-803 and currently in clinical trials). The IL15sa nanogel is a crosslinked, protein-polyethylene glycol (PEG) complex that breaks down slowly under physiological conditions allowing for pseudoautocrine stimulation of the CAR T cells with the IL15sa cargo. The effects of IL15sa nanogel loading on CAR T cell function were tested in a glioblastoma multiforme tumor model. Human T lymphocytes were lentivirally-transduced with a CAR targeting the epidermal growth factor variant III (EGFRvIII) antigen, as is currently being tested in early human clinical trials. EGFRvIII is expressed in up to 30% of glioblastoma multiforme cells but not on normal cells. EGFRvIII CAR T cells were loaded with a fluorophore-labeled IL15sa nanogel and examined in vitro for nanogel breakdown and effects on CAR T cell functions. The nanogel was detected on labeled cells for up to five days. Nanogels loaded with either control or IL-15sa did not alter antigen-specific cytolytic activity of EGFRvIII CAR T cells towards glioblastoma target cells. EGFRvIII CAR T cells loaded with control nanogel proliferated with the same kinetics as non-loaded CAR T cells upon co-culture with EGFRvIII+ glioblastoma targets. In contrast, IL15sa nanogel loaded CAR T cells had increased proliferation upon co-culture with antigenbearing targets. Additionally, IL15sa nanogel loaded EGFRvIII CAR T cells proliferated more with repeated stimulation of EGFRvIII-expressing glioblastoma cells. Studies are currently underway to test the efficacy of IL15sa nanogel-loaded EGFRvIII CAR T cells in vivo using a xenogeneic murine model of glioblastoma. These results indicate that nanogel loading of T cells can be a viable way of delivering immunostimulatory agents with CAR T cells. Nanogel loading of immune-modulating or tumoricidal drugs coupled to CAR T cells represents a novel platform for enhancing T cell function, and potentially for delivering systemically toxic drugs, into a specific and hostile tumor microenvironment.

IM-05 * MULTISPECIFIC CAR T CELLS FOR THE TREATMENT OF HIGH GRADE GLIOMA

BACKGROUND: High grade gliomas (HGG) carry a dismal prognosis. Our group has shown that patients' HER2-specific chimeric antigen receptor (CAR) T-cells kill autologous HGG, yet 40% of tumors recur in experimental animals. Recurrences are composed of HER2 negative escape variant cells that maintain positivity for two other glioma antigens, IL13Rα2 and EphA2. We hypothesized that co-targeting of these escape variants will enhance the anti-glioma activity of CAR T cells. OBJECTIVES: Objectives: The intent of this project is to develop a multi-specific T-cell product for the adoptive immunotherapy of HGG. DESIGN/METHOD: We generated and tested the effector functions of patients' T-cells co-expressing CARs for HER2, IL13Rα2 and EphA2 against autologous HGG cells derived from surgical excision samples. RESULTS: Primary patient HGG samples exhibited varied expression of the three target antigens, HER2, IL13Rα2 and EphA2. In order to render single patients' T cells tri-specific, we designed a transgene incorporating 3 encoding regions for HER2, IL13Rα2 and EphA2 CARs separated by 2A sequences in a single cassette driven by a CMV promoter. We used a retroviral system to stably integrate this transgene into the primary T cell genome. CAR-specific flow cytometry indicated proportionate stable expression of individual CAR molecules on the T cell surface. These T cells show distinct specificity for each of the three glioma antigens evidenced by activation, proliferation, and cytolytic function in immunoassays. Further, preliminary in vitro testing in glioma cell lines shows the ability to target populations of tumor cells that are not eliminated by unspecific products. CONCLUSION: The heterogeneity of antigenic expression in HGG and antigen escape justify targeting multiple glioma antigens simultaneously. We have successfully generated and tested a multi-specific T cell product for adoptive transfer that could offset antigen escape and exhibit an enhanced anti-glioma efficacy.

Chimeric antigen receptor-engineered T cells for the treatment of metastatic prostate cancer.

Cancer immunotherapy was selected as the Breakthrough of the Year 2013 by the editors of Science, in part because of the successful treatment of refractory hematological malignancies with adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells. Effective treatment of B cell leukemia may pave the road to future treatment of solid tumors, using similar approaches. The prostate expresses many unique proteins and, since the prostate gland is a dispensable organ, CAR T cells can potentially be used to target these tissue-specific antigens. However, the location and composition of prostate cancer metastases complicate the task of treating these tumors. It is therefore likely that more sophisticated CAR T cell approaches are going to be required for prostate metastasis than for B cell malignancies. Two main challenges that need to be resolved are how to increase the migration and infiltration of CAR T cells into prostate cancer bone metastases and how to counteract the immunosuppressive microenvironment found in bone lesions. Inclusion of homing (chemokine) receptors in CAR T cells may improve their recruitment to bone metastases, as may antibody-based combination therapies to normalize the tumor vasculature. Optimal activation of CAR T cells through the introduction of multiple costimulatory domains would help to overcome inhibitory signals from the tumor microenvironment. Likewise, combination therapy with checkpoint inhibitors that can reduce tumor immunosuppression may help improve efficacy. Other elegant approaches such as induced expression of immune stimulatory cytokines upon target recognition may also help to recruit other effector immune cells to metastatic sites. Although toxicities are difficult to predict in prostate cancer, severe on-target/off-tumor toxicities have been observed in clinical trials with use of CAR T cells against hematological malignancies; therefore, the choice of the target antigen is going to be crucial. This review focuses on different means of accomplishing maximal effectiveness of CAR T cell therapy for prostate cancer bone metastases while minimizing side effects and CAR T cell-associated toxicities. CAR T cell-based therapies for prostate cancer have the potential to be a therapy model for other solid tumors.

Interleukin-7 Mediates Selective Expansion of Tumor-redirected Cytotoxic T Lymphocytes (CTLs) without Enhancement of Regulatory T-cell Inhibition

The antitumor activity of chimeric antigen receptor (CAR)-redirected CTLs should be enhanced if it were possible to increase their proliferation and function after adoptive transfer without concomitantly increasing the proliferation and function of regulatory T cells (Treg). Here, we explored whether the lack of IL-7Rα in Treg can be exploited by the targeted manipulation of the interleukin-7 (IL-7) cytokine-cytokine receptor axis in CAR-engrafted Epstein-Barr Virus-specific CTLs (EBV-CTLs) to selectively augment their growth and antitumor activity even in the presence of Treg. We generated a bicistronic retroviral vector encoding a GD2-specific CAR and the IL-7Rα subunit, expressed the genes in EBV-CTLs, and assessed their capacity to control tumor growth in the presence of Treg in vitro and in vivo when exposed to either interleukin-2 (IL-2) or IL-7 in a neuroblastoma xenograft. We found that IL-7, in sharp contrast with IL-2, supports the proliferation and antitumor activity of IL-7Rα.CAR-GD2(+) EBV-CTLs both in vitro and in vivo even in the presence of fully functional Treg. IL-7 selectively favors the survival, proliferation, and effector function of IL-7Rα-transgenic/CAR-redirected EBV-CTLs in the presence of Treg both in vitro and in vivo. Thus, IL-7 can have a significant impact in sustaining expansion and persistence of adoptively CAR-redirected CTLs.

Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Significantly Mediate Burkitt Lymphoma (BL) Regression and Improve Survival In Human BL Xenografted NSG Mice

BackgroundBurkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma that occurs in children and adolescents (Miles/Cairo, BJH, 2012). The 5-year event-free survival (EFS) of children and adolescents with newly diagnosed BL has been significantly improved but for patients with relapsed/refractory disease, the prognosis is dismal due to chemotherapy resistance (Cairo, et al, J Clin Oncol, 2012; Cairo, et al Blood 2007). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. We have previously demonstrated that CD20 is expressing in ≥98% of pediatric BL (Perkins/Cairo, Clin Adv Hematol Oncol. 2003) and also reported that expanded Peripheral Blood Natural Killer (exPBNK) Cells electroporated with anti-CD20 Chimeric Antigen Receptor (CAR) mRNA have significant cytotoxicity against CD20+ BL in vitro (Chu/Cairo, et al, ASH, 2012). ObjectiveTo examine the anti-tumor effect of anti-CD20 chimeric antigen receptor (CAR+) modified expanded PBNK cells against disseminated CD20+ Burkitt Lymphoma in vivo using human CD20+ BL xenografted NSG mice. MethodsPBMC were expanded with lethally irradiated K562-mbIL15-41BBL cells for 14 days as we previously reported. CD56+CD3- expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit. CAR mRNA was nucleofected and CAR expression was detected using anti-mouse IgG, F(ab')2 fragment-specific antibody (Chu & Cairo, et al, ASH, 2012).5x105 Raji cells expressing luciferase (Raji-Luc) were intraperitoneally (i.p.) injected into the NSG mice (6 weeks, Jackson Labs). Raji engraftment and progression was evaluated using the Xenogen IVIS-200 system (Caliper Life Sciences) after i.p. injection of 150mg D-luciferin/kg/mouse. Photons emitted from luciferase-expression cells were quantified using the Living Image software. After Raji-Luc engraftment was verified in mice at day 7, 5x106 anti-CD20 CAR exPBNK or MOCK exPBNK (no anti-CD20 CAR mRNA electroporation) were i.p. injected to each mouse once a week (day 9,16, 23). Control mice received culture medium instead of NK cells. The cumulative luciferase signals were measured weekly to indicate the tumor growth, dissemination and progression.Statistical probability of survival and comparison of survival curves were determined by Kaplan Meier Method and the log-rank test. The results with a P value < 0.05 were deemed statistically significant. ResultsWe found that luciferase signals from Raji-luc were not significantly different between the untreated, mock exPBNK treated, and CAR exPBNK treated groups (p=0.0538) after the first NK injection. However, the luciferase signals measured in the CAR exPBNK treated group were significantly different than that in the control mice (two tailed P= 0.0013) and the mock exPBNK treated mice (two tailed P= 0.0229) after the second NK injection. After the third NK injection, the luciferase signals measured in the CAR exPBNK treated group were also significantly different than that in the control mice (two tailed P= 0.0173) and the mock exPBNK treated mice (two tailed P= 0.0256) (Fig. 1A). But luciferase signals were not significantly different between the untreated mice and the mock exPBNK treated mice (two tailed P= 0.0729).Consistent with the reduced luciferase signals, the CAR exPBNK treated mice had significantly extended survival time with median 40 days compared to the untreated mice (29 days, P<0.001) and the mock exPBNK treated mice (30 days, P<0.001) (Fig. 1B). There was no significant difference of survival between the untreated mice and the mock exPBNK treated mice. ConclusionMultiple injections of anti-CD20 CAR mRNA electroporated exPBNK cells can significantly mediate disseminated Raji tumor regression in Raji xenografted NSG mice compared to untreated and mock exPBNK treated mice. Furthermore, anti-CD20 CAR exPBNK significantly increased the survival of Raji xenografted mice. These results indicate therapeutic potential of multiple injections of anti-CD20 CAR mRNA modified exPBNK cells against BL in patients with chemo-immunotherapy resistance. Future directions include examining the anti-tumor activity of CAR+ exPBNK against rituximab/chemotherapy resistant BL in xenografted NSG mice. [Display omitted] Disclosures:No relevant conflicts of interest to declare.