Cell Aplasia Research Articles

Creation of the First Non-Human Primate Model That Faithfully Recapitulates Chimeric Antigen Receptor (CAR) T Cell-Mediated Cytokine Release Syndrome (CRS) and Neurologic Toxicity Following B Cell-Directed CAR-T Cell Therapy

The advent ofadoptive T-cell therapy using CD19 Chimeric Antigen Receptor (CAR) T cells has revolutionized the treatment of relapsed and refractory acute lymphoblastic leukemia (ALL). CAR T cells have shown encouraging results in clinical trials, with complete remissions in 90% of patients with refractory B-cell ALL. However, CD19 CAR T cell therapy is associated with significant side effects, including cytokine release syndrome (CRS), encompassing fevers, myalgias, hypotension, respiratory distress, coagulopathy as well as neurologic toxicity, ranging from headaches to hallucinations, aphasia, seizures and fatal cerebral edema. Our understanding of CRS and neurologic toxicity has been significantly limited by the lack of animal models that faithfully recapitulate these symptoms.We chose the non-human primate (NHP), Macaca mulatta, given that it closely recapitulates the human immune system, to create an animal model of B-cell-directed CAR T cell therapy targeting CD20. Rhesus macaques (n=3) were treated with 30-40mg/kg cyclophosphamide followed 3-6 days later by an infusion of CAR T cells at a dose of 1x107 transduced cells/kg. Recipient animals were monitored for clinical signs and symptoms of CRS and neurotoxicity, and data were collected longitudinally to determine CAR T cell expansion and persistence, B cell aplasia, as well as clinical labs of CRS and cytokine levels.Prior to testing the CD20 CAR T cells, we performed a control experiment, in which 1x107/kg control T cells, transduced to express GFP only (without a CAR construct), were infused following cyclophosphamide conditioning. This infusion resulted in short-lived persistence of the adoptive cellular therapy, with disappearance of the cells from the peripheral blood by Day +14 (Figure 1, green traces) and no clinical signs of CRS (Figure 2) or neurologic toxicities. In contrast, recipients of 1x107 cells/kg CD20 CAR-expressing T cells (n = 3) demonstrated significant expansion of the CAR T cells, and persistence for as long as 43days post-infusion, which corresponded to concurrent B cell aplasia (Figure 1). These recipients also developed clinical signs and symptoms of CRS as well as neurologic toxicity which was manifested by behavioral abnormalities and extremity tremors, beginning between days 5 to 7 following CAR T cell infusion, with the onset of clinical symptoms coinciding with maximum CAR T cell expansion and activation. The neurologic symptoms were responsive to treatment with the anti-epileptic medicationlevetiracetam. The clinical syndrome was accompanied by elevations in CRP, Ferritin, LDH and serum cytokines, including IL-6, IL-8 and ITAC (Figure 2 A and B), recapitulating data from clinical trials using CD19 CAR T cells. An expansion of CD20 CAR T cells on day 7 following infusion was also observed in the CSF in the animals, and coincided with the onset of neurotoxicity. Strikingly, we also detected CD20 CAR T cells in multiple regions of the brain via flow cytometry, including the frontal, parietal, and occipital lobes, as well as the cerebellum, and demonstrated an increased number of infiltrating T cells by immunofluorescence in the brains of animals treated with CD20 CAR T cells when compared to healthy controls.These data demonstrate the successful establishment of a large animal model of B-cell directed CAR T cell therapy that recapitulates the most significant toxicities of CAR T cell therapy, including CRS and neurotoxicity. This model will permit a detailed interrogation of the mechanisms driving these toxicities as well as the pre-clinical evaluation of therapies designed to prevent or abort them after CAR T cell infusion. [Display omitted] [Display omitted] DisclosuresKean:Juno Therapeutics, Inc: Research Funding. Jensen:Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Efficacy of Humanized CD19-Targeted Chimeric Antigen Receptor (CAR)-Modified T Cells in Children and Young Adults with Relapsed/Refractory Acute Lymphoblastic Leukemia

▪BackgroundTargeted immunotherapy with CTL019, CD19-specific chimeric antigen receptor (CAR)-modified T cells, can produce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). However, a subset of patients has limited persistence, which can increase the risk of relapse. Most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin; therefore, anti-mouse reactivity is one potential cause of immune-mediated rejection that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19-directed CAR T cells (CTL119).DesignA pilot/phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (CTL119) enrolled children and young adults with relapsed/refractory B-ALL with or without prior exposure to a CAR T cell product. Patients previously treated with CD19-specific CAR-modified T cells were eligible if they met 1 of 3 criteria: 1) CD19+ relapse 2) no response to prior CAR T cell therapy or 3) early B cell recovery indicating poor persistence of CAR T cells. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of CD3z, 4-1BB, and humanized anti-CD19 scFv domains and activated/expanded with anti-CD3/CD28 beads. The humanized scFv domain was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Patients received lymphodepletion with cyclophosphamide and fludarabine 1 week prior to infusion with CTL119.ResultsThirty children and young adults aged 29 mo-24 yr were infused with CTL119. Eighteen patients had received prior allogeneic stem cell transplant (SCT). Eleven patients who previously received murine-derived CD19-specific CAR-modified T cells (CTL019, n=7; other, n=4) were retreated for B cell recovery (n=5), CD19+ relapse (n=5), or no response to prior CAR T cells (n=1). CNS disease or other extramedullary disease was the indication for enrollment in 6 and 3 patients, respectively.At assessment 1 month after infusion, 26/30 patients (87%) achieved a complete response (CR), defined as morphologic remission with B cell aplasia. Of 11 patients previously treated with murine CD19-specific CAR-modified T cells, 7 (64%) achieved a CR at 1 month, 4 demonstrated no response. Multiparameter flow cytometry for minimal residual disease (MRD) was negative at a detection level of 0.01% in 5/7 responding patients. Two responding patients with positive MRD progressed to CD19+ relapse at 1.6 and 3 mo.In patients with no prior exposure to a CD19 CAR T cell product, MRD-negative CR was achieved in 19/19 patients (100%). One patient relapsed with CD19+ extramedullary disease at 2.8 mo. With a median follow-up of 4.2 mo (range, 1.0-14.1 mo) for all responding patients in both cohorts, 23/26 remain in remission with 1 proceeding to SCT in remission.B cell aplasia, a functional marker of CD19-targeted CAR T cell persistence, continued for 3 months or more in 11/18 patients with adequate follow-up: 1/6 retreatment, 10/12 CAR-naïve. Cytokine release syndrome (CRS) was observed in 28/30 patients and mild in most patients (grade 1, n=6; grade 2, n=18). Three patients experienced grade 3 CRS requiring supplemental oxygen or low-dose vasopressor support and 1 experienced grade 4 CRS requiring high-dose vasopressor and ventilatory support. Severe CRS was successfully managed with the IL6R antagonist tocilizumab in 3 patients. Neurologic toxicity included encephalopathy (n=5) and seizure (n=4) and was fully reversible.ConclusionIn the first study of humanized anti-CD19 CAR T cells, CTL119 induced remissions in children and young adults with relapsed/refractory B-ALL, including 64% of patients previously treated with murine CD19-directed CAR T cells and 100% of CAR-naïve patients. Further investigation into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. DisclosuresMaude:Novartis: Consultancy. Barrett:Novartis: Research Funding. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding; Vitality Institute: Research Funding. Brogdon:Novartis: Employment. Scholler:Novartis: Patents & Royalties: Royalties, Research Funding. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Frey:Amgen: Consultancy; Novartis: Research Funding. Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Employment. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership. Grupp:Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding.

Decreased Rates of Severe CRS Seen with Early Intervention Strategies for CD19 CAR-T Cell Toxicity Management

Introduction: CD19 CAR-T cell therapy is a promising strategy in the treatment of pre-B ALL, with early phase trials showing results of >90% CR rates. Over 90% of patients develop CRS concurrent with proliferation of CAR-T cells. Rates of severe CRS (sCRS) in previously reported studies have ranged from 23% to 43%. Concerns have been raised that the use of immunomodulation may impact engraftment and proliferation of CAR-T cells, potentially impairing efficacy. Additionally, concern has been raised that the use of tocilizumab (toci) may lead to an increased risk of neurotoxicity. Here we report our clinical experience with early treatment of CRS in a cohort of patients who received pre-emptive toci and dexamethasone (dex) with the goal to lessen the occurrence of sCRS.Methods: Subjects enrolled on the PLAT-02 study (NCT02028455) underwent apheresis and CAR-T cell manufacturing followed by lymphodepletion and CAR-T cell infusion. In this phase 1 dose escalation study, subjects were treated from 0.5- 10 x 106 CAR T cells/kg. sCRS included the use of pressors or inotropes. Severe neurotoxicity included any grade 3/4 neurotoxicity, excluding headache, and grade≥ 2 seizure. The first 23 subjects received toci (8mg/kg) with or without steroids for dose limiting toxicity (DLT). Subsequently the protocol was modified to provide guidelines for early intervention with CRS. The next 20 subjects received toci and subsequent dex (5-10mg every 6-12 hours prn) for persistent symptoms using clinical criteria, with the goal to prevent sCRS. Clinical criteria included persistent fever of ≥ 39°C despite antipyretics for 10 hours, persistent/recurrent hypotension after initial fluid bolus, and initiation of oxygen supplementation. Engraftment of CAR T cells and B cell aplasia was determined by flow cytometryResults: The two cohorts had similar overall rates of CRS: 91% (21/23) vs 95% (19/20), with higher rates of sCRS in the initial cohort: 30% (7/23) vs 15% (3/20) (p=0.3). Neurotoxicity was seen in 48% v 50% with similar rates of severe neurotoxicity, 22% v 25%. In the first cohort of subjects, 22% (5/23) received toci and 17% (4/23) received steroids due to DLT. In the second cohort, there was an increase in the number of subjects receiving toci to 50% (11/20, p=0.032) with an increase in steroid use as well to 30% (6/20, p=0.5)The overall rate of MRD-negative CR in this study was 93% (40/43) and this was not impacted by the use of toci or dex. The rate of MRD-negative CR in those subjects receiving toci without steroids, toci with steroids, and steroids alone were also similar (89% vs 100% vs 100%). Additionally the MRD-negative CR rate in the first cohort was similar to the early intervention cohort (91% v 95%). Continued peripheral blood expansion of CAR+ T cells can be seen in subjects who have received tocilizumab and/or steroids. There were no differences detected among the immunomodulatory groups with regards to peak percentage engraftment, area under the curve, or functional persistence of CAR T cells.Conclusions: Despite encouraging efficacy, the toxicity associated with CD19 CAR-T cell therapy gives rise to concerns about its widespread use. Early intervention with immunomodulation appears to decrease the rates of sCRS while preserving the high rates of MRD-negative CR. Additionally, the engraftment and persistence of CAR+T cells is not impacted by the use of toci and/or steroids when given early after the onset of clinical symptoms of CRS. Although not intended to assess the impact of toci on neurotoxicity, it is of note that the rates of neurotoxicity, and in particular severe neurotoxicity, did not increase. These results warrant further study of the impact of early immunomodulation for the prevention of sCRS. DisclosuresGardner:Amgen: Honoraria. Li:Juno Therapeutics: Employment, Equity Ownership. Jensen:Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia.

The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies.

Abstract 4893: Molecular detection of ALL in the peripheral blood is more sensitive than flow cytometric analysis of the bone marrow in patients with treatment-related hypocellularity

Abstract Measurable residual disease detection (MRD) is an important tool for early detection and monitoring relapse. Traditionally, bone marrow is collected and analyzed by flow cytometry (FC) to evaluate MRD status in acute lymphoblastic leukemia patients (ALL). More recently, highly sensitive molecular methods such as deep sequencing allow for higher sensitivity detection of MRD. Although the bone marrow is considered to be more representative of tumor burden than sampling the blood, bone marrow collection is more invasive, disruptive, and resource intensive than peripheral blood draws. ALL patients enrolled in the Seattle Children's Pediatric Leukemia Adoptive Therapy (PLAT-02) trial undergo MRD assessment pre and post CD19 CAR T cell infusion using FC analysis of a bone marrow sample. We have used high throughput sequencing (HTS) in paired blood and bone marrow samples from these patients to evaluate the complete B cell repertoire and assess MRD up to 63 days post-infusion. To-date, we have obtained 38 paired samples. Using HTS alone we have observed a significant correlation in both the frequency of total reads and the number of malignant cells per million in paired peripheral blood and bone marrow samples. 26/38 of the bone marrow samples were determined either aparticulate or hypocellular. Importantly, 8/14 of patients that were MRD negative in the bone marrow by FC were MRD positive in the blood by HTS. These data suggest, that, in cases where the bone marrow is hypocellular or aparticulate, peripheral blood samples evaluated by HTS are more sensitive than traditional flow cytometric analysis in the bone marrow for detection of MRD. The impact of B cell aplasia may be important in increasing the sensitivity of HTS in the detection of MRD. The ability to detect an ALL B cell clone in the blood through HTS has many clinical implications; blood can be sampled more often, is less of a burden to patients, and can provide earlier prediction of relapse. Citation Format: Hannah Smithers, Olivia Finney, Ilan Kirsch, Beryl Crossley, Michael Jensen, Rebecca Gardner. Molecular detection of ALL in the peripheral blood is more sensitive than flow cytometric analysis of the bone marrow in patients with treatment-related hypocellularity. [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 4893.

Biology and clinical application of CAR T cells for B cell malignancies.

Chimeric antigen receptor (CAR)-modified T cells have generated broad interest in oncology following a series of dramatic clinical successes in patients with chemorefractory B cell malignancies. CAR therapy now appears to be on the cusp of regulatory approval as a cell-based immunotherapy. We review here the T cell biology and cell engineering research that led to the development of second generation CARs, the selection of CD19 as a CAR target, and the preclinical studies in animal models that laid the foundation for clinical trials targeting CD19+ malignancies. We further summarize the status of CD19 CAR clinical therapy for non-Hodgkin lymphoma and B cell acute lymphoblastic leukemia, including their efficacy, toxicities (cytokine release syndrome, neurotoxicity and B cell aplasia) and current management in humans. We conclude with an overview of recent pre-clinical advances in CAR design that argues favorably for the advancement of CAR therapy to tackle other hematological malignancies as well as solid tumors.

277. Small Molecule-Regulated Antigen Recognition System for Inducible T Cell Targeting of Cancer Cells

Redirecting T cells against tumors by introducing chimeric antigen receptors (CAR) has demonstrated promising clinical results in certain cancers. However, the constitutive activity of these receptors limits which antigens can be targeted with this approach and may result in persistent elimination of healthy antigen-expressing cells. Successful CAR-T targeting of CD19+ tumors, for example, produces chronic B cell aplasia and necessitates life-long intravenous immunoglobulin (IVIG) treatment. Daric is an alternative antigen targeting approach that aims to: i) minimize the long-term toxicity of CAR-T treatment; ii) allows targeting of previously inaccessible antigens; and iii) is amenable to multiplex antigen targeting and other advanced targeting designs and strategies. The Daric system separates the antigen recognition and signaling functions of a CAR into two distinct polypeptides that contain the FKPB12 and FRB dimerization domains but lack signaling activity in the absence of a dimerization agent. Addition of the FKBP12-FRB bridging drug rapamycin or a non-immunosuppressive rapamycin analogue AP21967 heterodimerizes the signaling and antigen recognition components and restores signaling competency for antigen-dependent T cell activation. Importantly, the FKB12 and FRB dimerization partners are located extracellularly, minimizing interference with endogenously expressed signaling components and eliminating the requirement for rapamycin/AP21967 cell penetrance. A range of extracellular linkers and transmembrane domains were used to design a variety of CD19-targeting Daric constructs that exhibit minimal basal activity and robust drug-dependent antigen-specific cytolytic activity and cytokine production. The CD19-specific Daric T cells exhibited comparable levels of cytokine release, proliferation and cytolytic activity compared to a CD19-targeting CAR T cells. In vitro, Daric T cells were activated at sub-nanomolar to nanomolar concentrations of rapamycin or AP21967. No differences in cellular phenotype, expansion or functional responsiveness of Daric T cells compared to CAR T cells were observed. As expected, rapamycin was immunosuppressive to CAR T cell functionality, however Daric T cells continued to produce high level of cytokines even in the presence of rapamycin. These results highlight the potential of the Daric system to target highly expressed antigens and minimize the off-tumor on-target toxicity associated with traditional CAR designs.

Abstract IA13: Engineered T cells for cancer therapy

Abstract Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a limited survival. Chimeric antigen receptor (CAR)-modified T cells targeting CD19 may overcome the limitation of low complete response rates with conventional therapies with the potential to induce sustained remissions in patients with refractory B-cell malignancies. We previously reported preliminary results on three patients with refractory CLL. Here we report the mature results from our initial trial using CAR modified T cells to treat 14 patients with relapsed and refractory CLL (1). Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14–11 ×108 CTL019 cells (median 1.6 x 108 cells). Patients were monitored for toxicity, response, expansion and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8/14 (57%), with 4 complete remissions (CR), and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and CAR T cells have persisted and remain functional beyond 4 years in the first two patients achieving CR; no patient in CR has relapsed. All responding patients have developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease is not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL. In separate studies we have asked whether the same CD19 CAR may have activity in tumors that do not express CD19. A patient with refractory multiple myeloma was treated with CTL019 cells, a cellular therapy consisting of autologous T cells transduced with an anti-CD19 chimeric antigen receptor, following myeloablative chemotherapy (melphalan 140 mg/m2) and autologous stem cell transplantation (2). Four years earlier, autologous transplantation with a higher melphalan dose (200 mg/m2) had induced only a partial, transient response. Autologous transplantation followed by CTL019 cells led to a complete response with no evidence of progression and no measurable serum or urine monoclonal protein at last evaluation, 12 months after treatment. This response was achieved despite absence of CD19 expression in 99.95% of this patient's neoplastic plasma cells.

Efficacy and Safety of Humanized Chimeric Antigen Receptor (CAR)-Modified T Cells Targeting CD19 in Children with Relapsed/Refractory ALL

Background Targeted immunotherapy with T cells expressing a chimeric antigen receptor (CAR) can produce dramatic anti-tumor responses. We previously demonstrated complete remissions (CRs), prolonged persistence, and sustained responses in children and adults with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL) treated with CD19-specific CAR-modified T cells (CTL019). However, a subset (~25%) of patients has limited persistence of CTL019, which can increase the risk of relapse. We hypothesized that repeat infusion may prolong persistence in some patients but would be ineffective in patients with immune-mediated rejection. As most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin, anti-mouse reactivity is one potential cause of immunogenicity that may be overcome by fully human or humanized CAR design. We now report on retreatment with murine or humanized CD19-directed CAR T cells. Aims We sought to 1) prolong persistence by reinfusion of murine CTL019 in children with r/r CD19+ ALL and evidence of poor persistence and 2) determine the safety and efficacy of CD19-directed CAR T cells with a humanized scFv domain in children with B cell recovery or CD19+ relapse after prior CAR T cell therapy. Method T cells collected from patients were transduced with a lentiviral vector encoding a CAR composed of murine or humanized anti-CD19 scFv, CD3z, and 4-1BB domains, activated/expanded ex vivo with anti-CD3/anti-CD28 beads, cryopreserved, and then infused. The humanized scFv was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Results Of 50 patients in CR at 1 month after CTL019 infusion (50/53 CR), 14 patients received a repeat infusion of murine CTL019 at 3 and/or 6 months after initial infusion and 11 are evaluable for response. Indications were B cell recovery (n=4), CD19+ minimal residual disease (MRD, n=2), or undetectable CTL019 in peripheral blood by flow cytometry (≤0.1% CTL019+ of CD3+, n=5). Minimal toxicities were observed and included fever and fatigue. CTL019 reinfusion induced B cell aplasia for a second time in 1 of 4 children treated for B cell recovery. Two of these patients later relapsed, 1 with CD19+ and 1 with CD19- disease. Of 2 patients with CD19+ MRD, 1 had no response and subsequently experienced a CD19+ relapse and the second became MRD-negative but had B cell recovery. All 5 children reinfused for poor CTL019 persistence demonstrated continued B cell aplasia 3-15 months after repeat infusion with detectable CTL019 in peripheral blood by flow cytometry (0.2-2.8% CTL019+ of CD3+) in 4 patients. Of this group, 4 patients remain in remission 12-21 months after initial infusion, and 1 experienced a CD19- relapse. To overcome the potential for anti-murine immune-mediated rejection, patients previously treated with CAR-modified T cells were eligible for a phase 1 study of humanized CD19-directed CAR T cells (CTL119). Five children were infused for B cell recovery (n=3) or CD19+ relapse (n=2). Cytokine release syndrome (CRS), seen in 2 patients, was limited to fever, headache, and nausea and did not require vasopressor or respiratory support. Three patients showed no response and progressed (n=1) or relapsed with CD19+ (n=1) or CD19- (n=1) ALL. Responses were demonstrated in 2 patients: 1) MRD-negative CR in a patient treated for CD19+ relapse and 2) reduction in MRD and return of B cell aplasia in a patient treated for CD19+ MRD and B cell recovery, with subsequent progression to CD19+ relapse. Both responding patients were previously resistant to murine CTL019 reinfusion, suggesting a possible immune-mediated rejection mechanism. Ongoing studies are investigating anti-CAR responses. Conclusion Repeat infusion of murine CTL019 may prolong B cell aplasia in patients with early evidence of poor persistence and in a fraction of patients with B cell recovery. In the first demonstration of humanized anti-CD19 CAR efficacy, retreatment with CTL119 expressing a humanized scFv induced remission of ALL refractory to prior CD19-directed murine CAR T cell therapy, suggesting that immune-mediated rejection may be an important mechanism of resistance in the subset of patients with rapid CAR cell loss and loss of B cell aplasia. Further investigation into anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy, Research Funding. Off Label Use: CTL019 and CTL119 for relapsed/refractory ALL. Rheingold:Endo: Other: Husband's employer, has equity interest; Novartis: Consultancy. Aplenc:Sigma Tau: Consultancy. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding. Brogdon:Novartis: Employment. Loew:Novartis: Employment. Zheng:Novartis: Patents & Royalties. Levine:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding.

Latest in Clinical Application of CAR Cell Therapy for B-cell Malignancy and Transplantation

Unparalleled remission rates in patients with chemorefractory B-ALL treated with CD19-CAR T cells illustrate the potential for immunotherapy to eradicate chemoresistant cancer. CD19-CAR therapy is poised to fundamentally alter the clinical approach to relapsed B-ALL and ultimately may be incorporated into frontline therapy. Despite these successes, as clinical experience with this novel modality has increased, so has understanding of factors that limit success of CD19-CAR T cells for leukemia. These insights have implications for the future of cell based immunotherapy for leukemia and provide a glimpse of more global challenges likely to face the emerging field of cancer immunotherapy. Five challenges limiting the overall effectiveness of CD19-CAR therapy will be discussed: 1) T cell exhaustion is a differentiation pathway that occurs in T cells subjected to excessive T cell receptor signaling. A progressive functional decline occurs, manifest first by diminished proliferative potential and cytokine production, following by diminished cytolytic function and ultimately cell death. High leukemic burdens predispose CD19-CAR T cells to exhaustion as does the presence of a CD28 costimulatory signal, while a 4-1BB costimulatory signal diminishes the susceptibility to exhaustion. This biology is likely responsible for limited CD19-CAR persistence observed in clinical trials using a CD19-zeta-28 CAR compared to that observed using a CD19-zeta-BB CAR. 2) Leukemia resistance occurs in approximately 20% of patients treated with CD19-CAR and is associated with selection of B-ALL cells lacking CD19 targeted by the chimeric receptor. Emerging data demonstrates two distinct biologies associated with CD19-epitope loss. Isoform switch is characterized by an increase in CD19 isoforms specifically lacking exon 2, which binds the scFvs incorporated into CD19-CARs currently in clinical trials. Lineage switch is characterized by a global change in leukemia cell phenotype, and is associated with dedifferentiation toward a more stem-like, or myeloid leukemia in the setting of CD19-CAR for B-ALL. These insights raise the prospect that effectiveness of immunotherapy for leukemia may be significantly enhanced by targeting of more than one leukemia antigen. 3) CAR immunogenicity describes immune responses induced in the host that can lead to rejection of the CD19-CAR transduced T cells. Anti-CAR immune responses have been observed by several groups, and mapping is underway to identify the most immunogenic regions of the CAR, as a first step toward preventing this complication. 4) The most common toxicities associated with CD19-CAR therapy are cytokine release syndrome, neurotoxicity and B cell aplasia. Cytokine release syndrome is primarily observed in the setting of high disease burdens and efforts are underway to standardize grading and treatment algorithms to diminish morbidity. Increased information is needed to better understand the neurotoxicity observed in the context of this therapy. Although clinical data is limited, B cell aplasia appears to be adequately treated with IVIG replacement therapy. 5) Technical graft failure (e.g. inadequate expansion/transduction) is a challenge that has received limited attention, primarily since many trials have not reported the percentage of patients in whom adequate products could not be generated. We have observed that technical graft failure is often associated with a high frequency of contaminating myeloid populations in the lymphocyte product and selection approaches designed to eradicate myeloid populations have resulted in improved T cell expansion and transduction. These results suggest that optimization of lymphocyte selection may diminish the incidence of technical graft failure. Disclosures Mackall: Juno: Patents & Royalties: CD22-CAR. Off Label Use: cyclophosphamide.

Durable Remissions in Children with Relapsed/Refractory ALL Treated with T Cells Engineered with a CD19-Targeted Chimeric Antigen Receptor (CTL019)

BACKGROUND CARs combine a targeting antibody (scFv) domain with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR, which have resulted in up to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 53 pts with relapsed/refractory (r/r) ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with r/r CD19+ ALL. 48/53 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 10 7 to 10 8 cells/kg with a transduction efficiency of 3.6-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, and not their allo donors. RESULTS We treated 53 children and young adults with CD19+ ALL, median age 11y, (4-24y). To assess disease burden after lymphodepleting chemotherapy, pts had BM aspirations performed 1D prior to 1 st CTL019 infusion: 41/53 pts had detectable ALL while 12 were MRD(-). A median of 4.3x10 6 CTL019 cells/kg (1-17.4x10 6 /kg) were infused over 1-2D (1 pt got cells over 3D). There were no infusional toxicities >gr2, although pts who developed fevers within 24h of infusion did not receive a planned 2 nd infusion of CTL019 cells. 50 pts (94%) achieved a CR, including a patient with CD19+ T ALL, 3 did not respond. MRD measured by clinical flow cytometry was All but 5 (90%) of pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 28% of patients and was reversed in all cases with the IL6-receptor antagonist tocilizumab, together with short courses of corticosteroids in 9 pts. Although T cells collected from the 35 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells can be detected by flow cytometry and/or QPCR, and results in the pharmacodynamic marker of CTL019 function, B cell aplasia, which continued for 3-39 months after infusion in pts with ongoing responses. B cell aplasia has been treated with IVIg without significant infectious complications. CONCLUSIONS: CTL019 cells can undergo robust in vivo expansion and can persist for 3 years or longer in children and young adults with r/r ALL, allowing for the possibility of long-term disease control without subsequent therapy such as SCT. This approach also has promise as salvage therapy for patients who relapse after allo SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with r/r ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CTL019 resistance. Rapid loss of CTL019 cells (prior to 3 months) is associated with a high risk of CD19+ relapse. CTL019 therapy has received Breakthrough Therapy designation from the FDA in pediatric and adult ALL, and phase 2 multicenter registration trials are well underway. Disclosures Grupp: Novartis: Consultancy, Research Funding. Maude: Novartis: Consultancy, Research Funding. Shaw: Novartis: Research Funding. Aplenc: Sigma Tau: Consultancy. Lacey: Novartis: Research Funding. Levine: Novartis: Patents & Royalties, Research Funding. Melenhorst: Novartis: Research Funding. Rheingold: Novartis: Consultancy; Endo: Other: Husband9s employer, has equity interest. Teachey: Novartis: Research Funding. Wood: Novartis Pharmaceuticals Corporation: Employment. Porter: Novartis: Other: IP interest, Research Funding; Genentech: Other: Spouse employment. June: University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding.

Choice of Better T-cells for Adoptive Immunotherapy

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. The definition of optimal targets and antigen receptors as well as the differentiation status of transferred T cells are emerging as crucial parameters for generating cell products with predictable efficacy and safety profiles. Our laboratory has demonstrated that defined subsets within the memory CD8+ T cell compartment fulfill all key characteristics of adult tissue stem cells and are essential for robust and long-term maintained responses upon adoptive transfer. We have developed clinical multi-parameter enrichment technologies to purify these memory stem cells for clinical applications. In my presentation I will report on the status of ongoing clinical trials using such purified cell products either as a primary T cell population for the treatment of infections upon allogeneic stem cell transplantation or after genetic modification with a CD19 CAR for the treatment of malignancies (collaboration with Stan Riddell, FHCC/Seattle). Infusing small numbers of T cells within a memory stem cell product can be highly effective therapeutically, but bears some risk of toxicity. Therefore, safeguards that allow selective depletion of transferred cells in the case of un-tolerable side effects may be needed to further improve adoptive immunotherapy. I will present results exploring the capacity of a truncated version of EGFR (EGFRt) co-expressed with T cells expressing a CD19-CAR. In pre-clinical mouse models we demonstrate that application of Cetuximab, which binds to EGFRt, confers selective depletion of adoptively transferred CAR-T cells in vivo. Long-term B cell aplasia, which is a main side effect of CD19-CAR T cell therapy, can be completely reverted with this strategy. Vaccination studies upon B cell recovery demonstrate full functionality of antigen-specific antibody formation. EGFRt co-expressing CD19-CAR T cells have been successfully transferred into first human patients, providing the option to test for the first time in a clinical setting whether treatment of B cell aplasia after long-term leukemia remission can be achieved by selective depletion. DisclosuresBusch:STAGE cell therapeutics: Other: I was share holder of STAGE cell therapeutics, a company that was recently bought by Juno therapeutics.. Off Label Use: CD19 CAR T cells.

A Novel Strategy of Switching on/Off CD19CAR Expression Under Tetracycline-Based System

Introduction: Genetic modification of T cells with chimeric antigen receptor (CAR) has emerged with astonishing treatment outcomes for B cell malignancies. Clinical trials of CAR-T therapy demonstrated toxicities such as hypogammaglobulinemia due to B cell aplasia or hemophagocytic syndrome after overactivation of CAR-T cells. These toxicities are considered as major drawbacks for broader application of CAR-T therapy. To overcome these serious adverse events, further modification of CAR-T technology to control CAR expression arbitrary is needed. Therefore we aimed to develop inducible CAR expressing T cells based on tetracycline-regulation system.Methods: We developed a novel inducible CD19CAR system by infusing anti-CD19-CD3z-CD28-tEGFR into pRetroX-TetOne vector (Tet-19CAR). By using Tet-19CAR transduced SUPT1 (T cell line), expression and disappearance kinetics of CAR were determined. We also retrovirally transduced Tet-19CAR into human CD8+ T cells, and achieved more than 90% purity of CAR positive T cells after a selection with anti-EGFR mAb. These CAR-T cells were again expanded with anti-CD3/28 beads and used in 51 Cr release assay, coculture assay, cytokine release assay and T cell proliferation assay. Regarding coculture assay, CD19 transduced K562-CD19 (K562-CD19) was labeled with 0.1 nM CFSE and plated with CAR-T cells at a ratio of 1:1 without IL-2 supplementation and incubated for 96 hours. Finally we examined this system in NOG mice. We injected 0.5 x 106 Raji-ffluc (fire-fly luciferase) followed by 5.0 x 106 CAR-T cells from the tail vein, then we evaluated the tumor flux by in vivo imaging system on days 7, 14, 21, and 30.Results: With more than 100 ng/mL of Doxycycline (Dox), CD19CAR was successfully expressed on both of SUPT1 and CD8+ T cells. For maximum and minimum expression, 24 and 72 hours were needed after addition and discontinuation of Dox, respectively. To determine the cytotoxicity of Tet-19CAR-T cells according to presence or absence of Dox, we performed 51 Cr release assay and coculture assay against K562-CD19. In the presence of Dox, Tet-19CAR showed an equivalent lytic activity to conventional CD19CAR-T cells (c19CAR). In contrast, Tet-19CAR without Dox exhibited significantly lower cytotoxicity against CD19+ target cells. (Dox (-) Tet-19CAR, Dox (+) Tet-19CAR and c19CAR: 14.0±4.0%, 38.0±4.0% and 37.0±2.0% at an E:T ratio = 10:1, respectively). In the coculture assay, Tet-19CAR with Dox eradiated K562-CD19, while they failed to suppress the target cells without Dox.In the intracellular IFN-g assay against K562-CD19, a similar proportion of responder was IFN-g + in Tet-19CAR with Dox and c19CAR. On the other hand, a significantly low proportion of IFN-g + cells were observed in Tet-19CAR without Dox. (Dox (-) Tet-19CAR, 1.0%±0%, Dox (+) Tet-19CAR, 19.1%±6.0% and c19CAR 21.5%±4.0%, respectively)Similar to intracellular IFN-g assay, ELISA revealed that Tet-19CAR with Dox and c19CAR produced IL-2 and IFN-g equally well. However, Tet-19CAR without Dox hardly did. [IL-2 (ng/ml): Dox (-) Tet-19CAR, 1.00±0.060, Dox (+) Tet-19CAR, 9.25±0.30 and c19CAR 8.75±0.68; IFN-g (ng/ml): 2.32±1.24, 57.96±6.95 and 62.42±5.95] (Fig).We next analyzed CAR-T cell proliferation upon stimulation with K562-CD19 over 96 hours. Tet-19CAR with Dox showed 6-7 fold expansion, whereas Tet-19CAR without Dox failed to proliferate.Regarding in vivo model, the mice treated with c19CAR or Tet-19CAR with Dox showed significantly low tumor flux but the mice treated with Tet-19CAR without Dox showed higher tumor burden at day 21 of CAR-T cell infusion [Photons/sec: Dox (-) Tet-19CAR, 2.5 x 1010, Dox (+) Tet-19CAR, 6.4 x 108 and c19CAR, 8.4 x 108 ].Conclusions: We generated tetracycline-inducible CAR-T cells and successfully controlled the CAR expression with Dox administration. Tet-19CAR without Dox still demonstrated some CD19CAR expression and subsequent cytotoxicity against CD19 positive cells. Nonetheless the CAR expression level of Tet-19CAR without Dox was lower than the threshold for exhibiting positive responses in the function assays such as cytokine production and proliferation. This phenomenon was also confirmed in the xenograft model. To regulate CAR expression more precisely and pursue clinical translations in combinations with other CARs, further efforts are needed to reduce any leaky CAR expression by modification of the system. [Display omitted] DisclosuresKiyoi:Pfizer Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

Impacts of ABO incompatibility on early outcome after single unit unrelated cord blood transplantation: a retrospective single center experience

目的研究供受者ABO血型不合对单份非血缘脐血造血干细胞移植（UCBT）患者中性粒细胞、血小板、红细胞植入，急性移植物抗宿主病（aGVHD）累积发生率及180 d移植相关死亡率（TRM）的影响。方法以2008年4月至2014年10月行单份UCBT的208例患者为研究对象，其中供受者ABO血型相合99例，次要不合60例，主要不合38例，主次均不合11例。全部患者均采用强化清髓预处理方案，并予环孢素联合霉酚酸酯方案预防GVHD。结果ABO血型相合与不合的UCBT相比，中性粒细胞、血小板、红细胞累积植入率，Ⅱ~Ⅳ度aGVHD、Ⅲ~Ⅳ度aGVHD及180 d TRM的累积发生率差异均无统计学意义（P值均>0.05），且无一例发生纯红细胞再生障碍。A供O血型不合患者中性粒细胞、血小板、红细胞的中位植入时间分别为+17（+12~+28）、+37（+17~+57）、+25（+13~+52）d，Ⅱ~Ⅳ度aGVHD、Ⅲ~Ⅳ度aGVHD及180 d TRM累积发生率分别为37.50%（95%CI 34.39%~40.61%）、6.25%（95%CI 5.48%~7.02%）及23.53%（95%CI 21.30%~25.76%）。与ABO血型相合、次要不合及主次均不合组比较，差异均无统计学意义（P值均>0.05）。结论接受ABO血型不合UCBT的患者移植后无纯红细胞再生障碍发生。供受者ABO血型不合对UCBT患者的造血重建、aGVHD发生率及TRM均无影响。

Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.

Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. Chimeric antigen receptor (CAR)-modified T cells targeting CD19 have the potential to improve on the low complete response rates with conventional therapies by inducing sustained remissions in patients with refractory B cell malignancies. We previously reported preliminary results on three patients with refractory CLL. We report the mature results from our initial trial using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL. Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14 × 10(8) to 11 × 10(8) CTL019 cells (median, 1.6 × 10(8) cells). Patients were monitored for toxicity, response, expansion, and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8 of 14 (57%), with 4 complete remissions (CR) and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and the CAR T cells persisted and remained functional beyond 4 years in the first two patients achieving CR. No patient in CR has relapsed. All responding patients developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease was not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL.

Abstract IA10: CARs for leukemia and beyond?

Abstract Relapsed hematologic malignancies pose a substantial unmet therapeutic challenge despite aggressive therapies. Engineered T cells have shown promise for a variety of malignancies and chronic infections. In this session we will review the current status of trials testing chimeric antigen receptor (CAR)-modified T cells for acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML) and for myeloma. As of April 2014, twenty-five children and 5 adults with relapsed or refractory ALL were treated with anti-CD19:4-1BB:CD3zeta CAR T cells (CTL019). Complete remissions (CR) were achieved in 27/30 patients (90%). CTL019 cells proliferated up to 1000-fold and were detectable in blood, bone marrow, and cerebrospinal fluid of responding patients. Sustained remissions were achieved in 15/22 evaluable patients with median follow-up of 7 months (2-24 months), with event-free survival of 67% (95% confidence interval [CI], 51% to 88%) and overall survival of 78% (95% CI, 65% to 95%) measured at 6 months. CTL019-mediated B cell aplasia persisted for up to 2 years and is ongoing. Cytokine release syndrome (CRS) was observed in all patients. Severe CRS, seen in 27% of patients, was associated with high disease burden and was effectively treated with the IL-6 receptor antibody tocilizumab. More than 50 patients with advanced refractory chronic lymphocytic leukemia have been treated with CTL019. Two of the first three patients treated remain in complete remission nearly 4 years after infusion. An ongoing randomized phase II trial testing a high and a low dose of CTL019 has not shown a difference in the response rate. The lack of an obvious dose response rate is consistent with the high level proliferation of CAR T cells in vivo and suggests that once a threshold dose is reached, further dose escalation does not increase efficacy. Persistent B cell aplasia is observed in responding patients (more than 3 years in CLL patients and more than 2 years in pediatric ALL patients). No patient with persistent B cell aplasia has relapsed with CD19+ tumor. A major issue in the field is whether it is necessary to have ongoing immunosurveillance by CAR T cells or whether they induce “sterile remissions” where every last tumor cell is eradicated. CAR T cells can target very low levels of targets expressed at the cell surface. Because some myeloma cells express low levels of CD19, and because B cells may be a precursor of malignant myeloma cells, an ongoing pilot trial is testing whether CTL019 cells might recognize and eliminate malignant cells in patients with advanced plasma cell myeloma. In preclinical studies we have found that CD33 and CD123 CAR T cells have potent therapeutic effects in mice bearing xenografts with AML. At issue is whether off tumor toxicity on normal cells will be tolerable with CD33 and CD123 specific CARs. In summary, CAR-modified T cell therapy directed against CD19 is highly effective as salvage therapy for children and adults with relapsed/refractory ALL and CLL. Moreover, long-term remissions may be possible in some patients. Our findings support the advancement of CAR-modified T cell therapy into multicenter phase 2 clinical trials. 1. M. Kalos, C. H. June, Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39, 49 (2013). 2. S. A. Grupp et al., Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. New England Journal of Medicine 368, 1509 (2013). 3. D. L. Porter, B. L. Levine, M. Kalos, A. Bagg, C. H. June, Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia New England Journal of Medicine 365, 725 (2011). 4. M. Kalos et al., T cells expressing chimeric receptors establish memory and potent antitumor effects in patients with advanced leukemia. Science Translational Medicine 3, 95ra73 (2011). 5. S. Maude et al., Sustained Remissions with Chimeric Antigen Receptor T Cells for Leukemia. New England Journal of Medicine, in press (2014). Citation Format: Carl H. June, David Porter, Stephan Grupp, Bruce L. Levine. CARs for leukemia and beyond? [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA10.

T Cells Engineered with a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Have Long Term Persistence and Induce Durable Remissions in Children with Relapsed, Refractory ALL

BACKGROUNDCARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL.METHODST cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors.RESULTS30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. [Display omitted] All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion.Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONSCTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. DisclosuresGrupp:Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.

T Cell Products of Defined CD4:CD8 Composition and Prescribed Levels of CD19CAR/Egfrt Transgene Expression Mediate Regression of Acute Lymphoblastic Leukemia in the Setting of Post-Allohsct Relapse

Introduction. ALL relapse following allogeneic HSCT has dismal outcomes due in large part to ineffective therapies. The primary objectives of the Phase 1 portion of this study, which is restricted to the subset of pediatric and young adult patients who relapse following HSCT, is to determine the feasibility of manufacturing products of defined composition and transgene expression, the safety of the cryopreserved T cell product infusion, and to describe the full toxicity profile, including development of clinically significant GVHD. Therapeutic responses are also tracked based on multiparameter flow and IgH deep sequencing.Methods. CD4 and CD8 T cell subsets are immunomagnetically isolated from apheresis products obtained from the research participant. Following anti-CD3xCD28 bead stimulation, T cell lines are transduced with a SIN lentiviral vector that directs the co-expression of the FMC63scFv:IgG4hinge: CD28tm:4-1BB:ζ CAR and the selection/tracking/suicide construct EGFRt. Transduced cells are propagated using recombinant human cytokines to numbers suitable for clinical use over 10-20 days during which time they are subjected to EGFRt immunomagnetic positive selection. Shortly following lymphodepleting chemotherapy, cryopreserved CD4/EGFRt+ and CD8/EGFRt+T cell products are thawed and infused at the bedside such that patients receive a 1:1 ratio of EGFRt+CD4 and CD8 T cells at the protocol prescribed dose level.Results. Six subjects (4m – 3 yr s/p HSCT) have been treated at dose level 1 (5 X 105 CAR T cells/kg); four were treated with active disease, and two were treated while MRD negative. All patients received lymphodepleting chemotherapy prior to T cell infusion. The infusions were well tolerated with only 1 AE > grade 2 (grade 3 anaphylaxis related to the DMSO). All five responding subjects exhibited in vivo expansion of CAR T cells (peak engraftment 13.4 - 93.6 % CAR+ T cells/circulating T cells occurring 8-14 days post infusion) that were predominantly CD8+. Subjects with higher disease burden had higher peak PB CAR T cell levels compared to those with MRD negative marrows (62.7% v 19.6%). Accumulation of CAR T cells in bone marrow and CSF was observed. Five of the six subjects obtained or maintained an MRD negative CR following CAR T cell therapy, while the subject who did not have a therapeutic response failed to have detectable cell product engraftment. Four of the five subjects who had engraftment of CAR T cells have ongoing persistence with accompanying B cell aplasia and leukemia control (35 days-5 months as of August 1, 2014). Only one subject required immunomodulatory treatment for sCRS (tocilizumab and dexamethasone), and that subject lost persistence at day +42. Of the 2 subjects with identified malignant IgH rearrangement, deep sequencing of bone marrow (BM) showed no evidence of the clone by day +21 and day +63, respectively. Each of the five responding subjects developed some degree of CRS with fever and hypotension as the hallmark symptoms (2/6 admitted to ICU). Three of these patients developed encephalopathy (2 grade 1 and 1 grade 4) that was fully reversible. One subject developed de novo grade 2 acute skin GVHD shortly following CAR T cell engraftment and CRS. Skin biopsy revealed that only 9% of skin localized T cells marked EGFRt+ while 79% of circulating T cells marked EGFRt+. This subject was treated with a 2 week course of 1 mg/kg of prednisone, followed by a rapid taper over a six week period and resolution of the GVHD, and has ongoing persistent CAR+T cells.Conclusions. Infusions of 5x105 defined composition CD4:CD8 CD19CAR/EGFRt+ T cells/kg have produced encouraging rates of MRD negative CRs in pediatric and young adults ALL patients who have suffered a postHSCT relapse. Based on intent to treat, we have found it is feasible to generate donor-derived products from each of the six enrolled patients. Expected toxicities include CRS with ~30% ICU admission rate and encephalopathy with severity ranging from mild to severe, but fully reversible. Although one patient developed acute GVHD post T cell therapy, our preliminary assessment suggests that CAR T cells were not mediators of this response. This study continues to accrue at increasing dose levels and updated results will be reported at the meeting. DisclosuresOff Label Use: tociluzimab for treatment of cytokine release syndrome cetuximab for the ablation of T cells CAR T cells for the treatment of leukemia. Jensen:Juno: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Abstract PR06: T cells engineered with a chimeric antigen receptor (CAR) targeting CD19 (CTL019) produce complete responses and long-term persistence without GVHD in children with relapsed, refractory ALL

Abstract Background: CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains into a single chimeric protein. We previously reported on CTL019 cells expressing a CAR recognizing CD19 combined with the CD137 and CD3 zeta intracellular activation domain, including 1 sustained CR in a patient with ALL (Grupp, et al. NEJM 2013). We now report on outcomes and longer follow up from our pilot study treating 16 children with relapsed, refractory ALL. Methods: T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into pts with relapsed or refractory CD19+ ALL. 13/16 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency (TE) of 11-45%. 11 pts had relapsed ALL after a prior allogeneic SCT. T cells were collected from the pt, regardless of prior SCT status, though all pts had to be 6 mos s/p SCT with no GVHD or GVHD treatment. Results: 16 children median age 9.5 y (5-22y) with CD19+ ALL were treated. One child had T cell ALL aberrantly expressing CD19. 14/16 pts had active disease or +MRD after chemotherapy on the day prior to CTL019 cell infusion, while 2 were MRD(-). A median of 4x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 3 pts developed fevers within 24 hrs of infusion and did not receive planned subsequent infusions of CTL019 cells. 13 patients (81%) achieved a CR, including the patient with CD19+ T ALL, 3 did not respond. 10/16 evaluable pts as of August 28, 2013 have ongoing BM CR with median follow up 3.4 mo (1.2-16 mo). Three patients with a CR at 1 month have subsequently relapsed, 1 with CD19(-) disease. All responding pts developed some degree of delayed cytokine release syndrome (CRS) at the time of peak T cell expansion, and a subset experienced transient hypotension and hypoxia. Treatment with tocilizumab, an anti IL6-receptor antagonist, reversed the CRS related hemodynamic or respiratory instability seen, though corticosteroids were used concurrently in some patients. No GVHD was observed, and cells collected from SCT recipients were generally of 100% donor origin. Persistence of CTL019 cells detected by flow cytometry and/or QPCR in pts with ongoing responses continued for 1-15 months after infusion, resulting in complete B cell aplasia during the period of CTL019 persistence. Pts have been treated with IVIg without any unusual infectious complications. One child who entered a CR subsequently developed MDS with a new trisomy 8 in ALL remission, and 1 child developed a single leukemia cutis lesion at 6 mo, still BM MRD(-). Conclusions: CTL019 cells are T cells genetically engineered to express an anti-CD19 scFv coupled to CD3ζ signaling and 4-1BB costimulatory domains that can undergo robust in-vivo expansion and can persist for 15 mo or longer in pts with relapsed ALL. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anticytokine treatment. This approach has promise as a salvage therapy for patients who relapse after allo-SCT, and collection of tolerized cells from the recipient appears to have a low risk of GVHD. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL. This abstract is also presented as Poster B69. Citation Format: Stephan A. Grupp, Shannon Maude, Richard Aplenc, David M. Barrett, Michael Kalos, Bruce Levine, Manuel Lichtman, Susan Rheingold, Angela Shen, Christine Strait, David Teachey, Patricia A. Wood, Carl June. T cells engineered with a chimeric antigen receptor (CAR) targeting CD19 (CTL019) produce complete responses and long-term persistence without GVHD in children with relapsed, refractory ALL. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR06.

Long-Term Functional Persistence, B Cell Aplasia and Anti-Leukemia Efficacy In Refractory B Cell Malignancies Following T Cell Immunotherapy Using CAR-Redirected T Cells Targeting CD19

▪ BackgroundRecent advances in T cell engineering have enabled clinical trials to evaluate the potential for adoptive transfer of T cells to target malignancy. A single treatment with engineered gene-modified T cells has the potential to generate potent and long-lasting anti-tumor immunity. We have reported initial clinical data on the use of T cells engineered to express a Chimeric Antigen Receptor (CAR) that targets CD19 (CTL019) in patients with advanced treatment-refractory CLL and pediatric ALL (Porter, et al NEJM 2011; Kalos et al. Sci Trans Med 2011, Grupp et al. NEJM 2013). The initial cohort of patients is now disease free between 1 and 3 years post-infusion. In this report we present data on the functional persistence, trafficking, and bioactivity of CTL019 cells from the initial cohort of CLL patients, a larger and more recently treated cohort of CLL patients, a cohort of pediatric ALL patients, as well as an initial cohort of adult ALL patients. MethodsCAR engineering of patient T cells was accomplished by lentivirus transduction followed by in-vitro expansion using anti-CD3 and anti-CD28 antibody-coated beads. Persistence of gene-modified T cells was assessed by quantitative PCR. CAR19 surface expression was detected by flow. B cell aplasia was evaluated by multiparametric flow cytometry. Multiplex cytokine analyses were performed using LuminexTMtechnology. ResultsDetailed clinical outcomes for each patient cohort will be reported separately at this meeting (Porter, D.L.et al, Grupp S. et al). In summary, as of July 15, 2013: For CLL: A total of 24 adult patients with advanced relapsed and/or treatment-refractory CLL have been treated under two separate protocols. To date 5 patients have achieved ongoing CR, 7 patients PR and 12 patients were NR at the primary endpoint (within 3 months post infusion). For ALL (pediatric): A total of 14 pediatric patients with treatment refractory ALL have been treated and were evaluable under a single protocol. To date 8 patients have ongoing CR, 4 patients have relapsed including 2 with CD19-negative disease and 2 patients were NR. For ALL (Adult): A total of 3 adult patients with advanced relapsed and/or treatment-refractory ALL have been treated under a single protocol. All 3 patients have ongoing CR; one patient went into an allogeneic transplant while in CTL019-induced CR.In all patients with CR, robust in vivo expansion of CTL019 cells was observed, as assessed by both molecular and flow cytometric analysis, followed by contraction and in all but one patient ongoing stable persistence of engineered cells, elimination of tumor B cells and ongoing B cell aplasia in blood and marrow at all evaluated time points (minimum 3 months, maximum ongoing at 35 months). In CR patients, peak marking exceeded 5% of total CD3+. Patients with PR demonstrated less robust in-vivo expansion accompanied by transient B cell elimination, while NR patients demonstrated minimal in-vivo expansion.Clinical MRD analysis in patients was supplemented with Illumina-HiSeq/MiSeq-deep sequencing based molecular MRD analysis. These analyses demonstrate that CRs were accompanied with deep and sustained molecular remissions as assessed by the absence of the tumor specific clonotype defined in the enrollment samples.All patients in CR experienced on-target cytokine release syndrome (CRS) and macrophage activation syndrome (MAS). Multiplex-cytokine analysis of serum samples from patients in CR demonstrated a broad pro-inflammatory signature with significant elevation in a subset of soluble immune modulators including IFN-g, IL-6, and IL2ra. In contrast, patients with NR did not have elevated serum cytokines. Elevation of cytokines coincided with expansion of CTL019 cells, elimination of B cells, and toxicity suggesting the potential for a cytokine-based diagnostic signature to monitor CTL019 treatment efficacy. ConclusionsAdoptive transfer of CTL019 cells engineered to express CD137 and TCR-zeta signaling domains can result in in-vivo expansion, homing to marrow, and long-term functional persistence of engineered cells, accompanied by ongoing complete clinical responses and long-term B cell aplasia in a substantial fraction of patients with advanced, refractory and high risk CLL and relapsed refractory pre- B cell ALL. These results highlight the potential of CTL019 therapy to effectively target CD19-positive malignancy. Disclosures:Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. Zheng:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Grupp:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding.