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With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

On the basis of promising preclinical data demonstrating the eradication of systemic B-cell malignancies by CD19-targeted T lymphocytes in vivo in severe combined immunodeficient-beige mouse models, we are launching phase I clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia. We present here the validation of the bioprocess which we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semiclosed culture system using the Wave Bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in severe combined immunodeficient-beige mice bearing disseminated tumors. The validation requirements in terms of T-cell expansion, T-cell transduction with the 1928z CAR, biologic activity, quality control testing, and release criteria were met for all 4 validation runs using apheresis products from patients with CLL. Additionally, after expansion of the T cells, the diversity of the skewed Vbeta T-cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemorefractory CLL and in patients with relapsed acute lymphoblastic leukemia. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any CAR or T-cell receptor.

The use of plasmid DNA as vaccines for the treatment of cancer and infectious diseases is on the rise. In order to facilitate the manufacture of clinical-grade plasmid DNA for Phase I clinical trials, we developed a process whereby >200 mg plasmid could be produced in a single production run under Good Manufacturing Practices. A dedicated cleanroom (Class 10,000 with Class 100 biosafety cabinet) is utilized for production of the bacterial cell bank, fermentation, harvest/lysis of the biomass, and downstream purification. Fermentation requires three 16-18 h runs (approximately 12 L each) in shaker-flasks, yielding approximately 60 g bacterial paste following batch centrifugation. The biomass is alkaline-lysed, pooled, and the resulting flocculent precipitate is separated by a novel vacuum step, followed by depth-filtration. Downstream processing includes anion-exchange chromatography, utilizing Qiagen silica-based resin, and precipitation with isopropanol. Following precipitation, the DNA is harvested by centrifugation, dried, formulated, and sterile-filtered using a Sartorius Sartobran 150 filter prior to Final-Filling. All processing steps utilize sterilized, single-use components. This process results in a product manufactured according to regulatory guidelines. The plasmid DNA is sterile with >or=95% supercoiled DNA, an A260/A280 ratio>or=1.9, undetectable or extremely low residual endotoxin, RNA, genomic DNA, protein, and antibiotic. Residual solvent levels are negligible. The product yields the predicted profile upon restriction-enzyme digestion, is biologically active upon transfection and remains stable for several years at -20 degrees C. We have therefore developed a reproducible and cost effective process to manufacture clinical-grade plasmid DNA. This process can be adapted by other academic centers for human or large animal clinical trials.

Dietary vitamin C is essential for humans, primates, guinea pigs, and several other animals and insects that lack l-gulono-γ-lactone oxidase, the final enzyme in its biosynthetic pathway from glucose (25). Under physiological conditions, vitamin C predominantly exists in its reduced form, ascorbic acid (AA); it also exists in trace quantities in the oxidized form, dehydroascorbic acid (DHA). There are two known mechanisms for transporting vitamin C (21). A universal system, present in all cells, transports vitamin C as DHA via facilitative glucose transporters (34). Once inside the cell, DHA is rapidly reduced and accumulates as AA (34, 35). The second transport system is functional in specialized cells where AA is directly transported into cells via sodium-dependent AA cotransporters (SVCT1 and/or SVCT2) (33). AA functions as a cofactor for enzymes involved in the biosynthesis of collagen (26), carnitine (28), and norepinephrine (18) and in the amidation of hormones (8). In plasma and cells, AA is a powerful antioxidant, quenching reactive oxygen species (ROS) and reactive nitrogen species (10, 14). Intracellular vitamin C can prevent cell death and inhibit mutations induced by oxidative stress (12, 22, 37). During the process of quenching free radicals, ascorbate donates an electron, becoming the unstable intermediate ascorbyl radical that can be reversibly reduced back to ascorbate. Ascorbyl radical can donate a second electron and be converted to DHA (13, 14). DHA may be reduced back to AA or be irreversibly hydrolyzed to 2,3-diketo-gulonic acid, which then is metabolized to threonic and oxalic acid (14). In cells loaded with AA and exposed to hydrogen peroxide, AA is converted to DHA, some of which effluxes from the cells via the glucose transporters, thereby providing a mechanism for recycling vitamin C to the extracellular medium (12). Alternatively, intracellular DHA can be transported to intracellular compartments and organelles (2, 20). DHA functions primarily as a readily transportable form of vitamin C (36). ROS play a key role in cellular responses as chemical second messenger molecules, and conversely, antioxidants modulate selected signaling responses (24). For example, ROS activate transcription factors, such as NF-κB, that are important in host defense, inflammation, and apoptosis (1, 11, 32). Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), hydrogen peroxide, and ceramide, activate NF-κB by inducing the phosphorylation of IκB proteins (11, 19). Phosphorylated IκBα releases NF-κB and is itself degraded via proteasomal pathways (17), while unphosphorylated IκB associates with NF-κB in the cytosol, preventing its nuclear migration. It was initially reported that AA inhibits TNF-α-induced NF-κB activation in endothelial cells via activation of p38 mitogen-activated protein kinase (MAPK) (4); however, we recently showed that AA suppresses TNF-α-dependent activation of NF-κB by inhibiting the activation of kinases involved in the phosphorylation of IκBα (6). We investigated the modulation of NF-κB activation by vitamin C and found that DHA directly inhibited the kinase activity of IKKβ and IKKα in vitro and in cellular assays. Thus, our data suggest a dual mechanisms of action of vitamin C in regulating NF-κB function. First, as an antioxidant quenching ROS, AA inhibits ROS-mediated signaling events. Second, after oxidization to DHA, vitamin C directly inhibits IKK kinase activity.

Basement membrane matrix proteins are known to up-regulate granulocyte–macrophage colony-stimulating factor (GM-CSF) signaling in neutrophils and mononuclear phagocytes, but the mechanisms involved are poorly understood. We used the intracellular portion of the α subunit of the GM-CSF receptor (αGMR) to search for interacting proteins and identified the 67-kDa laminin receptor (LR), a nonintegrin matrix protein receptor expressed in several types of host defense cells and certain tumors, as a binding partner. LR was found to interact with the β subunit of the GMR (βGMR) as well. Whereas GM-CSF functions by engaging the αGMR and βGMR into receptor complexes, LR inhibited GM-CSF-induced receptor complex formation. Laminin and fibronectin binding to LR was found to prevent the binding of βGMR to LR and relieved the LR inhibition of GMR. These findings provide a mechanistic basis for enhancing host defense cell responsiveness to GM-CSF at transendothelial migration sites while suppressing it in circulation.

Extracellular stimuli signal for activation of the transcription factor NFkappaB, leading to gene expression regulating processes involved in immune responses, inflammation, and cell survival. Tumor necrosis factor-alpha (TNFalpha) activates NFkappaB via a well-defined kinase pathways involving NFkappaB-inducing kinase (NIK), which activates downstream multisubunit IkappaB kinases (IKK). IKK in turn phosphorylates IkappaB, the central regulator of NFkappaB function. We found that intracellular vitamin C inhibits TNFalpha-induced activation of NFkappaB in human cell lines (HeLa, monocytic U937, myeloid leukemia HL-60, and breast MCF7) and primary endothelial cells (HUVEC) in a dose-dependent manner. Vitamin C is an important antioxidant, and most cells accumulate ascorbic acid (AA) intracellularly by transporting the oxidized form of the vitamin, dehydroascorbic acid (DHA). Because ascorbic acid is a strong pro-oxidant in the presence of transition metals in vitro, we loaded cells with vitamin C by incubating them with DHA. Vitamin C-loaded cells showed significantly decreased TNFalpha-induced nuclear translocation of NFkappaB, NFkappaB-dependent reporter transcription, and IkappaBalpha phosphorylation. Our data point to a mechanism of vitamin C suppression of NFkappaB activation by inhibiting TNFalpha-induced activation of NIK and IKKbeta kinases independent of p38 MAP kinase. These results suggest that intracellular vitamin C can influence inflammatory, neoplastic, and apoptotic processes via inhibition of NFkappaB activation.

Vitamin C is present in the cytosol as ascorbic acid, functioning primarily as a cofactor for enzymatic reactions and as an antioxidant to scavenge free radicals. Human granulocyte macrophage–colony-stimulating factor (GM-CSF) induces an increase in reactive oxygen species (ROS) and uses ROS for some signaling functions. We therefore investigated the effect of vitamin C on GM-CSF–mediated responses. Loading U937 cells with vitamin C decreased intracellular levels of ROS and inhibited the production of ROS induced by GM-CSF. Vitamin C suppressed GM-CSF–dependent phosphorylation of the signal transducer and activator of transcription 5 (Stat-5) and mitogen-activated protein (MAP) kinase (Erk1 and Erk2) in a dose-dependent manner as was phosphorylation of MAP kinase induced by both interleukin 3 (IL-3) and GM-CSF in HL-60 cells. In 293T cells transfected with alpha and beta GM-CSF receptor subunits (αGMR and βGMR), GM-CSF–induced phosphorylation of βGMR and Jak-2 activation was suppressed by vitamin C loading. GM-CSF–mediated transcriptional activation of a luciferase reporter construct containing STAT-binding sites was also inhibited by vitamin C. These results substantiate the importance of ROS in GM-CSF signaling and indicate a role for vitamin C in downmodulating GM-CSF signaling responses. Our findings point to vitamin C as a regulator of cytokine redox-signal transduction in host defense cells and a possible role in controlling inflammatory responses.

We report on 16 patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) that we treated with autologous T cells expressing the 19-28z chimeric antigen receptor (CAR) specific to the CD19 antigen. The overall complete response rate was 88%, which allowed us to transition most of these patients to a standard-of-care allogeneic hematopoietic stem cell transplant (allo-SCT). This therapy was as effective in high-risk patients with Philadelphia chromosome-positive (Ph(+)) disease as in those with relapsed disease after previous allo-SCT. Through systematic analysis of clinical data and serum cytokine levels over the first 21 days after T cell infusion, we have defined diagnostic criteria for a severe cytokine release syndrome (sCRS), with the goal of better identifying the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the sCRS. Additionally, we found that serum C-reactive protein, a readily available laboratory study, can serve as a reliable indicator for the severity of the CRS. Together, our data provide strong support for conducting a multicenter phase 2 study to further evaluate 19-28z CAR T cells in B-ALL and a road map for patient management at centers now contemplating the use of CAR T cell therapy.

Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD(+) disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD(-) complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell-mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.

Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising strategy to treat patients with cancer. Apheresis products are commonly used as starting material for large-scale T cell selection using CD3/28 magnetic beads. At the time of collection, the composition in various cell lineages is highly variable. We have encountered manufacturing challenges with apheresis products containing high proportion of CD14+ cells. As an illustration, we investigated the transduction and expansion of T cells using an apheresis product derived from a patient with ALL and containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from a cryopreserved apheresis product using CD3/28 magnetic beads. A short adherence procedure decreased the CD14+ cell composition from 70% to 45%. On day 3, 350×106 vs 20 ×106 total cells were recovered from each group without and with the depletion step, respectively. Selected cells from both groups were transduced using the same dilution of vector stocks at a cell concentration of either 0.4×106/mL or 0.2×106/mL in tissue culture bags. We observed a drastic difference in transduction efficiency between the group without depletion (4.5%) and the group in which the CD14+ cells were depleted (38%). Cells from the depleted group expanded 350 fold from day 3 to day 10 (100% CD3+). Our results suggest that high CD14+ cell content poses a manufacturing challenge when the selection of the CD3+ cells in the apheresis product is performed with CD3/CD28 magnetic beads. The CD14+ cell threshold for successful manufacturing without depletion will be discussed and additional samples will be analyzed.

Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising emerging strategy to treat cancer patients. Large-scale manufacturing of cGMP-grade CAR T cells using patient T cells selected and activated by CTS™ Dynabeads® CD3/CD28 (Dynabeads) followed by transduction with retroviral vectors is being used in the context of many clinical trials by our laboratory and others. Although we have established a robust protocol using Dynabeads, it is important to explore alternative sources to pre-empt supply chain limitations of this critical reagent. To this end, we evaluated T cell activation with either Miltenyi TransAct CD3/28 (TransAct) beads or Miltenyi ExpAct Treg (ExpAct) beads. In small-scale experiments, PBMCs were directly activated with TransAct or ExpAct beads and compared with our standard T cell selection and activation using Dynabeads. Overall, the transduction efficiency and expansion of T cells were comparable upon activation with all three reagents. The TransAct bead-stimulated cells exhibited comparable effector memory (EM)/central memory (CM) phenotype to that of the Dynabeads stimulated cells. In line with the EM/CM phenotype, CAR T cells stimulated with either TransAct or Dynabeads and tranduced with CD19-targeted CAR demonstrated robust and comparable antitumor activity in a systemic NSG/CD19+ NALM6 tumor mouse model. We further tested the efficacy of TransAct beads using positively or negatively selected T cells in a large-scale cGMP grade CAR-T cell manufacturing setting. Both the transduction efficiency and expansion of selected CD3+ cells activated with TransAct beads and Dynabeads were comparable. CD19-targeted CAR T cells activated by either TransAct or Dynabead were subjected to an in vivo stress test by using decreasing amount of CAR-T cells to treat systemic CD19+NALM6 tumors in NSG mice. In this experimental setting, T cells stimulated with TransAct beads demonstrated equivalent if not better anti-tumor activity than T cells stimulated with Dynabeads. In conclusion, our pre-clinical results suggest that TransAct beads support efficient transduction and expansion of CAR T cells. TransAct activated T cells exhibit antitumor activity equivalent to Dynabeads activated T cells in our NSG/CD19+NAML6 stress test. Therefore, Miltenyi TransAct beads can be used as an alternative to Dynabeads to stimulate T cells in clinical trials aiming at evaluating CAR T cell safety and antitumor activity.

Reactive oxygen species (ROS) are key components of postreceptor intracellular signaling pathways; however, the role of ROS in signal initiation is uncertain. We discovered that receptor–ligand interaction caused the generation of hydrogen peroxide (H 2 O 2 ). Using members of the hematopoietin receptor superfamily, as well as EGF receptor, we show that H 2 O 2 is generated by specific receptor–ligand interaction in cells and in cell-free systems. With cognate ligand, the extracellular domain of the receptor was sufficient for H 2 O 2 generation. We also found that production of H 2 O 2 was diminished in a granulocyte–macrophage colony-stimulating factor receptor mutant unable to bind ligand. Exogenously added H 2 O 2 induced signaling in the absence of ligand, whereas catalase and a membrane-bound peroxiredoxin inhibited ligand-dependent signaling. Our results suggest that H 2 O 2 produced by receptor–ligand interaction is involved as a chemical mediator that facilitates cell signaling.

Chimeric antigen receptors (CARs) are artificial receptors for antigen that redirect antigen specificity, activate T cells and further enhance T cell function through their costimulatory component. Three groups, including our own, have reported objective tumor responses when infusing autologous T cells genetically modified with CD19-targeted CARs into patients with chronic lymphocytic leukemia (CLL), other indolent non-Hodgkin lymphomas (NHL) and, most dramatically in patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL). Here we report on 16 patients with relapsed or refractory B-ALL that we treated with autologous T cells expressing the 19-28z CAR specific to the CD19 antigen. The overall complete response rate was 88%, as assessed by morphological criteria and IgH deep sequencing. This allowed us to transition a majority of these patients to a standard of care allogeneic hematopoietic stem cell transplant (allo-SCT). This therapy was as effective in high-risk patients with Ph+ disease as in those with relapsed disease following prior allo-SCT. Through systematic analysis of clinical data and serum cytokine levels over the first 21 days following T cell infusion, we have defined diagnostic criteria for a severe cytokine release syndrome (sCRS), with the goal of better identifying the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the CRS. Additionally, we found that serum C-reactive protein (CRP), a readily available laboratory study, can serve as a reliable indicator for the severity of the CRS and a road map for patient management at centers now contemplating the use of CAR T cell therapy. Based on these remarkably robust clinical results and the toxicity management algorithm we present here, we will soon open a multi-center Phase II clinical trial to further evaluate the efficacy of 19-28z CAR T cells and prospectively validate our proposed CRS monitoring and intervention guidelines in patients who are treated with CAR therapy. Citation Format: Michel Sadelain, Renier Brentjens, Marco Davila, Isabelle Riviere, Xiuyan Wang, Shirley Bartido, Jae Park, Diana Bouhassira, Kevin Curran, Stephen Chung, Jolanta Stefanski, Oriana Borquez-Ojeda, Sergio Giralt. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT102. doi:10.1158/1538-7445.AM2014-CT102

In order to optimize the transduction of CD34+ cells, CD34+ derived Langerhans cells (LCs) and T lymphocytes for upcoming clinical trials, we determined which medium is best suited for each cell type. Our aim is to identify the formulation providing the best cell proliferation, survival and the highest transduction efficiency (TE) while maintaining the biological activity of the cells. To this end, we harvested and screened PG13-derived retroviral vector stocks (VS) collected in various media formulations. High-titer VS were consistently obtained by controlling PG13 cell-plating densities, time of cell growth and vector harvests. We also periodically determined the titer of the frozen VS to determine how long these VS can be stored for clinical applications.

Vitamin C (ascorbic acid) is required for normal host defense and functions importantly in cellular redox systems. To define the interrelationship between human immunodeficiency virus (HIV) infection and vitamin C flux at the cellular level, we analyzed vitamin C uptake and its effects on virus production and cellular proliferation in HIV-infected and uninfected human lymphoid, myeloid, and mononuclear phagocyte cell lines. Chronic or acute infection of these cell lines by HIV-1 led to increased expression of glucose transporter 1, associated with increased transport and accumulation of vitamin C. Infected cells also showed increased transport of glucose analogs. Exposure to vitamin C had a complex effect on cell proliferation and viral production. Low concentrations of vitamin C increased or decreased cell proliferation depending on the cell line and either had no effect or caused increased viral production. Exposure to high concentrations of vitamin C preferentially decreased the proliferation and survival of the HIV-infected cells and caused decreased viral production. These findings indicate that HIV infection in lymphocytic, monocytic, and myeloid cell lines leads to increased expression of glucose transporter 1 and consequent increased cellular vitamin C uptake. High concentrations of vitamin C were preferentially toxic to HIV-infected host defense cell lines in vitro.

Although most adults diagnosed with B-ALL can be induced into a first complete remission (CR1 rates of approximately 80-90%), a majority will relapse and develop chemorefractory disease. Novel, non-chemotherapy based treatments are needed for this group of patients. We have developed a novel CD19-targeted T cell-based therapy for patients with B cell malignancies. We isolate T cells from patients with relapsed/refractory B-ALL and genetically modify them with a chimeric antigen receptor (CAR) construct, termed 19-28z, which comprises a CD19 binding domain fused to the signaling domains of the CD28 costimulatory receptor and the ζ chain of the CD3 complex. CD19 is a universal B cell antigen expressed on all normal and malignant B cells. Expression of the 19-28z CAR by a T cell promotes binding of the CD19 antigen and triggers cytotoxicity, cytokine release and proliferation upon engagement of CD19. We are conducting a Phase I clinical protocol in adults with relapsed/refractory B-ALL (NCT01044069). Enrolled patients are leukapheresed and then re-induced with salvage chemotherapy. A T cell infusion (3 x 106 19-28z CAR T cells/kg) is administered following conditioning chemotherapy. We now describe the results from this Phase I protocol. Thirteen adults have been treated to date: eleven were enrolled with relapsed/refractory disease, while the other 2 were enrolled and leukapheresed during CR1, but not treated until they relapsed. The age of the patients ranged from 23 to 74 with a median age of 42. Three of the 13 patients had Philadelphia-chromosome positive B-ALL, which is considered the most negative genetic risk factor for adults with B-ALL. We were able to achieve the required T cell dose, despite collecting patients with high blast counts or marked lymphopenias, in all but one patient. Seven of the 13 patients were infused with 19-28z CAR T cells while they had gross residual disease (>5 to 70% blasts in the BM). The remaining patients had MRD, detected by flow cytometry or deep sequencing, at the time of 19-28z CAR T cell infusion. Six patients developed toxicities including high-grade fevers (>40°C), hypotension, hypoxia, mental status changes, and seizures. These episodes ran for approximately one week before they were halted by treatment with steroids or tocilizumab. The other 7 patients did not experience toxicities. All patients completely recovered and were able to leave the hospital. The occurrence of toxicities correlated with tumor burden so that patients with gross residual disease (>5% blasts in BM) developed toxicities, while patients with MRD had no evidence of toxicities. Ten out of the 12 patients with detectable disease before T cell infusion developed MRD- responses such that 5 of the patients with gross residual disease (blasts > 5% in BM) became MRD- and 5 MRD+ patients became MRD-. The rapidity of the responses was quite remarkable with MRD- results obtained as early as 7 -14 days after T cell infusion. Furthermore, despite the poor predicted outcomes of relapsed Ph+ B-ALL we were able to get these patients MRD-, as detected by both deep sequencing for the IgH rearrangement and qPCR for the bcr-abl transcript. Conclusion The potent induction of MRD- responses and the reversibility of toxicities occurring in a subset of patients strongly support integrating this therapy in the modern paradigm for B-ALL therapy, by facilitating access to allogeneic-stem cell transplantation (SCT) for patients with relapsed B-ALL. Indeed, out of the 13 patients treated to date, 4 underwent an allo-SCT and 5 are being prepared for an allo-SCT. Of the remaining 4 patients, 1 is a non-responder (a patient with extramedullary disease, but no disease in the bone marrow), 1 relapsed (not eligible for allo-SCT), 1 is in CR2 (medically not eligible for allo-SCT), and 1 has not been evaluated for treatment response yet. Overall, the results from this Phase I protocol demonstrate that toxicities associated with this therapy are predictable and manageable. Furthermore, the remarkable MRD- re-induction rate and facilitation for allo-SCT we report here warrant further evaluation of this therapy in a Phase II protocol. Disclosures: No relevant conflicts of interest to declare.

Background Patient T cells may be genetically modified to express chimeric antigen receptors (CARs) targeted to antigens expressed on tumor cells. We have previously reported initial results from a phase I clinical trial treating patients with chemotherapy refractory and relapsed CLL with autologous T cells modified to express the 19-28z CAR targeted to the CD19 antigen expressed on most B cell malignancies (Brentjens RJ et al., Blood 2011). While the anti-tumor activity of the CD19-targeted CAR-modified (CAR+) T cells has been modest in the setting of rapidly progressive and chemotherapy refractory disease, we have observed significantly increased rates of durable responses in CLL patients with reduced disease burden and chemotherapy-sensitive disease (Park JP et al., ASH Abstract 2011). In order to address the previously recognized limitation of CAR+ T cells in the setting of advanced disease, we designed a phase I clinical trial wherein previously untreated CLL patients with residual disease following the first-line chemotherapy will receive the CD19-targeted CAR+ T cells as a consolidative therapy. Patients and Methods Patients with previously untreated CLL with high-risk disease features as defined by the presence of unmutated IgHV, del11q or del17p received the first-line therapy consisting of 6 cycles of pentostatin, cyclophosphamide and rituximab (PCR). Patients who have achieved either partial response (PR) or complete response (CR) with detectable minimal residual disease (MRD) were enrolled to the trial and underwent leukapheresis. Autologous T cells collected by leukapheresis were transduced with a retroviral vector encoding the anti-CD19 scFv linked to CD28 co-stimulatory and CD3ζ signaling domains. Patients received cyclophosphamide conditioning therapy followed two days later by the infusion of the CAR+ T cells in 3 dose-escalating cohorts. Response assessment was performed according to the criteria established by the NCI-WG. Serial bone marrow aspirate and blood samples were assessed for the modified T cell persistence (assessed by flow and RT-PCR) and cytokine profile analysis Results To date, 8 patients have been enrolled and 6 patients received the CAR+ T cells, completing the two dose cohorts of 3x106 and 1x107 CAR+ T cells/kg (3 in each cohort). The median age was 61.5 years (range, 45 – 68). 6 pts had unmutated IgHV and 2 pts had del11q. Median follow-up from the time of T cell infusion was 7 months (range, 2 – 12 mos) at the time of this report, and the median time from the completion of the upfront PCR chemotherapy to the T cell infusion was 6.5 mos (range, 4 – 12 mos). No DLT was observed. Cytokine release syndrome (CRS) was observed in 2 patients as manifested by fever, nausea, anorexia and transient hypotension, and none required steroid or other anti-inflammatory agents. There was a positive correlation between the development of CRS and the modified T cell persistence. 2 patients who had PR following the first-line PCR chemotherapy achieved CR after the T cell infusion; 2 patients maintained PR; and 2 patients had progressive disease (PD). Of the 2 patients with PD, one achieved PR after 6 cycles of PCR but had progressive lymphocytosis with ALC doubling time of 1 month at the time of T cell infusion and the other patient relapsed in lymph node only while the marrow remained MRD negative. Conclusions The infusion of autologous CD19-targeted CAR+ T cells appears to be safe and has demonstrated promising anti-tumor efficacy to further improve CR rates in patients with high-risk CLL undergoing the first-line purine analog-based chemotherapy. While the number of treated patients is too small to draw a definitive conclusion, our findings suggest that the second-generation CD19-targeted CAR+ T cells has enhanced anti-tumor activity in patients with reduced disease burden and is more effective in eradicating disease in the marrow versus lymph nodes. Enrollment is ongoing to this study, and updated clinical and correlative data will be presented. Disclosures: No relevant conflicts of interest to declare.

Abstract 3566 B cell acute lymphoblastic leukemia (B-ALL) in adults has a dismal prognosis. Intensified, combinatorial chemotherapy yields remarkable results in pediatric ALL but less so in adults. As allogeneic hematopoietic stem cell transplantation (HSCT) is curative in only a minority of patients, there remains a dire need for effective therapies in this patient population. Building on our results obtained with genetically targeted T cells in xenogeneic models of ALL and our experience in patients with chronic lymphocytic leukemia (Brentjens, Rivière, Blood, 2011), we have recently initiated a clinical trial (NCT01044069) treating adults with relapsed or refractory B-ALL with autologous T cells expressing the 19–28z chimeric antigen receptor (CAR), a dual CD3zeta/CD28 signaling receptor specific for the B cell antigen CD19. Human peripheral blood T cells retrovirally transduced to express the 19–28z receptor specifically lyse normal and malignant B cells in vitro and eradicate established tumors in vivo in pre-clinical animal models. We report the results from the initial cohort of patients treated on this protocol. The first two patients had relapsed disease that achieved morphologic remission following re-induction chemotherapy. Following adoptive therapy with CAR-modified T cells, one patient achieved a B cell aplasia and molecular remission documented by deep sequencing, which demonstrated the disappearance of IgH rearrangements associated with the malignant clone. The other patient had achieved a molecular remission after re-induction chemotherapy but further developed a complete B cell aplasia following modified T cell infusion. While in molecular remission, both patients successfully underwent allogeneic HSCT and were therefore removed from the study. In contrast to these two patients, the third patient failed to achieve a morphologic remission after re-induction chemotherapy and had >60% blasts in the bone marrow (BM) at the time of T cell infusion. Within 12 hours of completing T cell infusion, the patient developed high-grade fevers, hypotension, and rigors. Serum analyses demonstrated a sharp rise in cytokines (IFNg, TNFa, IL6, IL2, and IL8), reflecting rapid and robust onset of T cell activation. High-dose corticosteroids initiated 5 days after T cell infusion controlled these symptoms. Post-T cell BM aspirate on day 8 demonstrated undetectable blasts. Flow cytometry could not detect blasts or B lineage cells, but readily identified 19–28z CAR+ T cells. Significantly, the patient's B-ALL tumor cells harbored a unique 9p21 cytogenetic deletion that was detected by FISH in 28% of nuclei immediately prior to T cell infusion and was reduced to 5% 8 days after T cell infusion. The next BM aspirate, performed on day 24 after T cell infusion, demonstrated a persistent morphologic remission, a recovering BM, and complete absence of any detectable 9p21 deletions. Furthermore, PCR amplification for IgH rearrangements in the BM 24 days after T cell infusion confirmed a molecular remission and a B cell aplasia. Collectively, the clinical outcomes from this initial cohort of 3 patients demonstrate for the first time the in vivo efficacy of CD19-targeted T cells to induce clinical and molecular remissions as well as B cell aplasia in adults with relapsed or refractory B-ALL. These early results strongly support further investigation of 19–28z targeted T cells to treat leukemias and suggest this is a potential salvage therapy for patients with relapsed/refractory B-ALL who are failing chemotherapy or are ineligible for allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

TPS2619 Background: Complete remission (CR) rates for B-ALL in adults is high (>80%), but overall survival remains low (approximately 30%). Patients with relapsed disease have a very poor prognosis with a median survival measured in months. Therefore, there is a dire need for novel therapies for adults with relapsed or minimal residual B-ALL. To this end we have developed a novel T cell therapy for B cell malignancies using patient derived T cells genetically modified to express the 19-28z chimeric antigen receptor (CAR), an artificial T cell receptor specific to the CD19 antigen expressed on most B cell tumors. Human T cells retrovirally modified to express the 19-28z CAR successfully targets and eradicates B cell tumors in vitro and systemic human B cell cancers in mice. We have recently published the results of our initial Phase I clinical trial experience (Brentjens, Rivière et al., Blood 2011) utilizing this technology in adults with chronic lymphocytic leukemia. These results suggest clinical activity and demonstrate a persistence of 19-28z+ T cells that is inversely associated with tumor burden. These studies suggest that 19-28z+ T cells may be particularly effective in relapsed B-ALL because these tumors retain a degree of chemotherapy sensitivity, which will allow the infusion of T cells after salvage chemotherapy when patients have low tumor burdens. To our knowledge, this is the first clinical trial using autologous CD19 targeted T cells in adults with B-ALL. Methods: We are enrolling patients to a Phase I T cell dose escalation trial (NCT01044069). Adults with CD19+ B-ALL classified as a CR, relapsed, or refractory are eligible for enrollment. After patients are enrolled they are leukapheresed to obtain peripheral blood T cells, which are then retrovirally transduced to express the 19-28z CAR and expanded in our GMP gene transfer facility. Enrolled patients with relapsed or refractory B-ALL undergo a cycle of re-induction chemotherapy and conditioning cyclophosphamide followed by infusion of autologous 19-28z+ T cells. To date, 11 patients have been enrolled and 2 patients have been infused with 19-28z+ T cells.

TPS4700 Background: Based on our preclinical animal models, we initiated a phase I dose-escalating study to assess safety, dose requirement and targeting efficiency of genetically directed autologous human T cells targeted to Prostate Specific Membrane Antigen (PSMA). Our approach is based on the infusion of autologous PSMA-targeted T cells utilizing the P28z second generation chimeric antigen receptor (CAR) in patients (pts) with metastatic CRPC, following iv cyclophosphamide (Cy) (trial NCT01140373). For safety, the herpes simplex virus-1 thymidine kinase (hsvtk) gene is co-expressed with the P28z receptor, and renders T cells sensitive to ganciclovir for immediate T cell elimination if needed. The expression of hsvtk enables PET imaging using radiolabeled FIAU to localize adoptively transferred T cells. The aims of the trial are to assess: (1) safety of PSMA-targeted T cells; (2) biologic and anti-tumor effects; (3) T cell persistence at tumor site; and (4) immune response. Methods: Autologous T cells are activated from a leukapheresis product using anti-CD3/CD28 Dynabeads. Release criteria include mean vector copy number by Q-PCR and vector identity by Southern blot, absence of Replication Competent Retrovirus and residual Dynabeads. Pts will be treated at 3 dose levels from 107 to 108 CAR+ T cells/kg. Four patients have been enrolled; 3 have been treated with 300mg/m2 of Cy one day before infusion of 107 CAR+ T cells/kg. Pts underwent baseline and post treatment CT, bone and PET scans. Pts are followed weekly, then monthly with blood work including immune and vector sequence monitoring. Results: The first 3 pts within the first cohort were successfully treated without toxicity. Two had stable disease for greater than 6 months with the third patient having disease progression. There were no acute adverse events. Conclusion: We have established an ex vivo transduction, expansion and therapeutic protocol for the generation and testing of safe, clinical-grade, PSMA targeted T cells. Pts enrolled at the next dose level of 3 x 107 CAR+ T cells/kg will be assessed as described and by imaging the transduced T cell population using 18F-FIAU as a radiotracer. The data pertaining to the planned T cell imaging will be presented as well.

Abstract 4309 The NY-ESO-1 antigen is an attractive prototype cancer-testis (CT) antigen because it is expressed in a wide variety of tumors, including myeloma, melanoma, and synovial cell sarcoma, among others, but has restricted expression in normal tissues (testis and placenta). Based on our previous experience with 3T3-based artificial antigen presenting cells (AAPCs) in expanding influenza and CMV-specific T cells, we hypothesized that AAPCs expressing the NY-ESO-1 antigen and optimal costimulatory molecules would efficiently expand NY-ESO-1 specific T cells for potential use as adoptive immunotherapy. Accordingly, AAPCs were transduced to express the common MHC allele HLA-A*0201, beta2-microglobulin, and the full-length NY-ESO-1 protein, together with the T cell costimulatory ligands CD58, CD54, and CD80. These A2/NY-ESO-1 AAPCs expanded NY-ESO-1 specific T cells from normal donors after prolonged culture (4 weeks). In patients with melanoma who had detectable NY-ESO-1 antibodies, AAPC stimulation of PBMC expanded NY-ESO-1 antigen-specific CD8+ T cells approximately 3–4 logs in 3 weeks. These expanded T cells were functional in that they lysed peptide-pulsed targets (T2 cells) and native melanoma tumor targets (SK Mel 37). Expanded CD8+ T cells also produced interferon-gamma, MIP-1 beta, TNF-alpha and CD107a (but not IL-2) in response to antigen, indicating a polyfunctional response. We are currently performing scale-up experiments and pre-clinical validations of these AAPCs to embark on a clinical trial of adoptive cell therapy of NY-ESO1-specific T cells in patients with NY-ESO1-expressing tumors. Disclosures: No relevant conflicts of interest to declare.

3045 Background: We have developed a novel immunotherapy strategy based on the genetic modification of patient T cells to recognize the B cell-specific cellular antigen CD19, expressed on B cell tu...