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Chimeric antigen receptor (CAR)-modified T cells are being investigated in many settings, including classical Hodgkin lymphoma (cHL). The unique biology of cHL, characterized by scant Hodgkin and Reed-Sternberg (HRS) cells within an immunosuppressive tumor microenvironment (TME), may pose challenges for cellular therapies directly targeting antigens expressed on HRS cells. We hypothesized that eradicating CD19+ B cells within the TME and the putative circulating CD19+ HRS clonotypic cells using anti-CD19-directed CAR-modified T cells (CART19) may indirectly affect HRS cells, which do not express CD19. Here we describe our pilot trial using CART19 in patients with relapsed or refractory cHL. To limit potential toxicities, we used nonviral RNA CART19 cells, which are expected to express CAR protein for only a few days, as opposed to CART19 generated by viral vector transduction, which expand in vivo and retain CAR expression. All 5 enrolled patients underwent successful manufacturing of nonviral RNA CART19, and 4 were infused with protocol-specified cell dose. There were no severe toxicities. Responses were seen, but these were transient. To our knowledge, this is the first CART19 clinical trial to use nonviral RNA gene delivery. This trial was registered at www.clinicaltrials.gov as #NCT02277522 (adult) and #NCT02624258 (pediatric).

We recently demonstrated that sustained remission in 41 CLL patients treated with the CD19-specific, 4-1BB/CD3zeta-signaling chimeric antigen receptor (CAR19) T-cells correlated strongly with the expansion and persistence of the engineered T cells and that important pathways such as T cell exhaustion, glycolysis and T cell differentiation segregated responders from non-responders (Fraietta et al., 2018, Nature Medicine). We here report two advanced, chemotherapy-resistant CLL patients with the longest (7 years) follow-up on any trial of CART19 cells. Both patients had received five therapies before being treated at the University of Pennsylvania with autologous, murine CTL019 (tisagenlecleucel) cells for their CLL in 2010, receiving 1.1e9 and 1.4e7 CAR19+ T cells, respectively. Both patients have persistence of CAR-engineered T cells and both patients are still in remission as determined by flow cytometry and deep sequencing of IgH rearrangements for 5.5-7 years. Thus, the infused CAR-T cells have maintained these patients in deep molecular remission of their disease for the longest period of time that has been reported to date. To understand the fate of the infused CAR-T cells we determined the phenotype, function, and clonal nature of the persisting CTL019 cells. Flow cytometric CART19 cell analyses demonstrated that early during the anti-leukemia response, activated, HLA-DR-expressing CD8+ CAR-T cells rapidly expanded, followed by similarly activated CD4+ CAR-T cells. With tumor clearance the CAR-T cell population contracted, but an activated CD4+ CAR-T cell population was maintained and was still detectable at the last follow-up of 7 years. The CD8+ CAR-T cell pool remained present at low frequencies. Both populations had acquired and maintained an effector memory phenotype, a phenotype most consistent with active disease control. Furthermore, the analysis of the classical immune checkpoint inhibitory markers PD1, TIM3, LAG3, and CTLA4 showed that only PD1 was expressed from the earliest to the latest time point on >80% of all CAR-T cells, whereas LAG3 and TIM3 were expressed only early on but lost after tumor clearance. These data suggest that the initial tumor clearance was mediated by CD8+ CAR-T cells, but sustained by a CD4+ CAR-T cell population that still actively engages with target cells. To understand the clonal nature of these long-term persisting CAR-T cells we used two complementary methods: a) CAR T cells were sorted from post-infusion aliquots during the first two years for T cell receptor-beta deep-sequencing (TCR-seq); b) the CAR integration sites in the genome were sequenced in the infusion product and in circulating CAR-T cells. TCR-seq analysis of early post-infusion time points demonstrated that the circulating CAR-T cell populations consisted of hundreds to thousands of distinct clones which in patient 1 and 2 displayed clonal focusing by 21 and 1 month post-infusion, respectively, with some clones making up as much as 12% (patient 1) and 48% (patient 2) of the CAR-T cell repertoire. The analysis of clonotype sharing at the various time points via Morisita's overlap index analysis similarly showed repertoire stabilization late (21 months; patient 1) and early (1 month; patient 2) after infusion. Lastly, fate mapping of the infused CART19 cells via CAR integration site analysis in the infusion product until the latest time point indicated that the infusion products for both patients had a very diverse, non-clonal make-up, containing over 8,000 and 3,700 integration sites in patients 1 and 2, respectively. The higher degree of clonality in patient 2 but not 1 CAR-T cells as seen by TCR-seq was confirmed by integration site analysis, as was the sharing of CAR-T cell clones over time. Importantly, whereas the CAR integration site repertoire in patient 1 was diverse in the first two years, it stabilized and trended towards oligoclonality 21 months after infusion. Lastly, CAR integration site analysis revealed a high degree of clonal persistence, suggesting that tumor control and B cell aplasia were maintained by few, highly functional CD4+ CAR-T cell clones. In summary, we demonstrate that in both patients with the longest persistence of CAR-T cells reported thus far, early and late phases of the anti-CLL response are dominated by highly activated CD8+ and CD4+ CAR-T cells, respectively, largely comprised of a small number of persisting CD4+ CAR-T cell clones. Disclosures Melenhorst: Parker Institute for Cancer Immunotherapy: Research Funding; Incyte: Research Funding; Casi Pharmaceuticals: Consultancy; novartis: Patents & Royalties, Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. Lacey:Novartis Pharmaceuticals Corporation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Young:Novartis: Patents & Royalties, Research Funding. Siegel:Novartis: Research Funding. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Background: Cellular therapy using anti-CD19 autologous chimeric antigen receptor modified T (CART19) cells demonstrates promising outcomes in several hematologic malignancies of B-cell origin, but this therapy has not been studied in HL patients (pts). While neoplastic HL Reed-Sternberg (HRS) cells are considered CD19 negative, circulating CD19 positive clonal HRS cell precursors and CD19 positive reactive cells within the HRS tumor microenvironment represent potential therapeutic targets for CART19 in HL. Methods: We designed companion pediatric (NCT02624258) and adult (NCT02277522) open-label pilot studies to estimate the feasibility, safety, and efficacy of CART19 cell infusions in pts with relapsed/refractory HL lacking curative treatment options. To allow transient CD19 targeting and limit the window for acute toxicity and B cell aplasia, we used autologous T-cells electroporated with mRNA encoding chimeric anti-CD19 immunoreceptor scFv (RNA CART19) cells in lieu of permanently modified cells engineered by viral transduction. The scFv is derived from a murine monoclonal antibody. Following pheresis and manufacturing of RNA CART19 cells, pts undergo up to 6 infusions of 8x105-1.5x106 RNA CART19 cells/kg/dose for pts Results: To date, 5 pts have been enrolled and had RNA CART19 manufactured. Four pts were infused with RNA CART19 and are evaluable for toxicity/response. Characteristics of the 5 pts include: median age 24 years (range 21-42), 4 (80%) with stage IV, median number of previous therapies 5 (range 4-9), 4 (80%) had stem cell transplant (SCT): 3 had auto SCT, 1 had both auto and allo SCT. Three pts previously progressed on PD-1 inhibitor. All 5 pts underwent successful manufacturing of RNA CART19. One pt developed MDS prior to RNA CART19 infusions and was taken off study. Four patients who underwent RNA CART19 infusions were treated with cyclophosphamide as per protocol. The median number of infused CART19 cells/kg/dose was 1.5x106 (range 7.3x105 -1.5x106). Each patient (pt) received 6 infusions over 2 weeks. Using qRT-PCR, RNA CART19 was detected in 80% of peripheral blood samples drawn within 2 hours after each infusion (Figure 1). RNA CART19 was also detected in 20% of samples drawn immediately prior each infusion reflecting persistence of RNA CART19 from previous infusion at 48 or more hours ago. No pt had RNA CART19 detected by Day (D) 21. There were no study related deaths or grade (G) 3/4 non-hematologic toxicities. Most common G1/2 toxicities at least possibly related to the RNA CART19 therapy occurring in > 1 pt included transient headache in 3 and insomnia in 2. There was no evidence of cytokine release syndrome or significant elevation in cytokines at any point. The ORR at D28 was 50%: 1 complete (CR) and 1 partial response (PR). One pt had stable disease (SD) and one pt had progressive disease (PD). The CR pt (#3) was noted to have persistent RNA CART19 cells in 3 samples drawn at about 48 hours after the prior infusion compared with the PD pt (#1) who had no RNA CART19 detected by this time point on any measurement. In 2 pts who responded (#3 and #4), number of CD19-positive B-cells by flow cytometry as % of total leukocytes declined by 50% on D28 when compared to baseline. There was no evidence of B cell aplasia. The pt in CR progressed at 3 months and the pt in PR was taken off study. As part of routine clinical care, the 2 responding pts (#3 and #4) are currently in CR on PD-1 inhibitor (one underwent auto SCT). The pt with SD is in PR on lenalidomide and the pt with PD died of disease progression. Conclusion: Targeting CD19 positive cells with non-viral, RNA-electroporated, transiently expressed CART19 cells is a feasible and safe strategy in pts with relapsed/refractory HL. We saw encouraging responses, but these were short-lived. We are planning a study for HL pts utilizing virally transduced CART19 cells that are capable of in vivo expansion in combination with PD-1 inhibitors. Disclosures Svoboda: Merck: Research Funding; BMS: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Celgene: Research Funding; Kite: Consultancy; Pharmacyclics: Research Funding. Gill: Novartis Pharmaceuticals: Patents & Royalties, Research Funding. Grupp: Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. Mato: DTRM: Research Funding; AstraZeneca: Consultancy; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Regeneron: Research Funding; Acerta: Research Funding; Portola: Research Funding; Kite: Consultancy; AbbVie: Consultancy, Research Funding; Gilead Sciences, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Dwivedy Nasta: Takeda: Research Funding; Incyte: Research Funding; Immunogen: Research Funding. Landsburg: Takeda: Research Funding; Curis: Consultancy, Research Funding. Levine: GE Healthcare: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Novartis Pharmaceuticals Corporation: Patents & Royalties, Research Funding. Porter: Immunovative Therapies: Other: Member DSMB; Novartis: Honoraria, Patents & Royalties, Research Funding; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Servier: Honoraria, Other: Travel reimbursement; Incyte: Honoraria. June: Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding; WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Schuster: Genentech: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Research Funding; Nordic Nanovector: Consultancy; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Seattle Genetics: Consultancy; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Honoraria.

T cells bearing a second-generation anti-CD19 chimeric antigen receptor (CAR) induce complete remission in >90% of patients with acute lymphoblastic leukemia (ALL) at our institution. However, disease may recur and we recently identified two molecular mechanisms of relapse (PMID: 26516065). We here present a novel mechanism of antigen-negative relapse in a pediatric ALL patient. A 21 year-old male patient was in third relapse at the time of enrollment onto our CTL019 trial (ClinicalTrials.Gov #NCT01626495). The patient achieved an MRD-negative complete remission 1 month after CTL019 infusion but relapsed nine months later. Quantitative PCR analysis of the transgene and flow cytometry for CAR19 protein analysis showed the expected expansion of the CART cells followed by log-normal decay following disease eradication. At relapse, however, the transgene copy numbers had increased without a concomitant rise in CAR19 protein-expressing T cells. The CAR protein was found to be expressed by the now CD19-negative CD45dimCD10+CD3negCD22+ leukemia. Molecular analysis via next-generation immunoglobulin heavy chain sequencing (NGIS) of the apheresis product, used for CTL019 manufacturing, and relapse marrow at 9 months demonstrated clonal identity of the relapsed clone, which carried two rearranged IgH alleles. Sequencing of the CD19, CD21, CD81, and CD225 loci did not reveal any mutations. The analysis of lentiviral vector integration sites (LVIS) of the infusion product and post-infusion specimens showed the following: a) the infusion product carried over 15,000 unique integration sites; b) only 7 LVIS were shared between this sample and month 9 and 20 relapse specimens, none of which were near proto-oncogenes; c) the relapsed leukemia carried two LVIS, one on chromosome 10, >50 kb distal from neuropilin (NRP1) and the second in an intron of proprionyl coenzyme A carboxylase-A (PCCA). Flow cytometric and qRT-PCR analysis of leukemic cells in the apheresis and relapse showed that NRP1 levels were indistinguishable, suggesting that the lentiviral vector did not act as an enhancer for NRP1. Furthermore, qRT-PCR demonstrated that the lentiviral integration did not affect the gene expression levels of PCCA. Investigation into the origins of the leukemic CAR transduction event showed that the patient did not exhibit replication-competent lentivirus. However, NGIS analysis of infusion product revealed the leukemic clonotypes this sample, indicating that the gene transfer occurred during the manufacturing of the CTL019 cells. A retrospective analysis of 115 aphereses from ALL patients showed that the index patient had an unusually high disease burden in the apheresis product with 63% of all cells expressing CD19; at harvest, however, the CTL019 product consisted of 99.21% T cells, highlighting the purging effect of the CD19-specific T cells during manufacturing. NGIS analysis of infusion products of 17 additional ALL patients also identified the leukemic clonotype(s) in 6 more products. Only one additional patient demonstrated CAR19 protein expression on the leukemic cells, and this clone was not dominant at relapse (0.075% of all leukemic cells expressed the CAR). Our investigation into the biology of CAR19-expressing ALL cells showed the following: 1) the in vitro analysis of BBζ-signaling CAR19 showed no evidence of cytokine secretion; 2) the infusion of the baseline leukemia and CAR19-expressing leukemic cells from the same patient in mice did not demonstrate differential pharmacodynamics, even after restimulation with human CD19-expressing murine B cells in vivo; 3) the CD19 protein was detectable using flow cytometry and confocal microscopy, but only with an antibody recognizing an intracellular epitope; 4) importantly, the relapsed clone was indeed resistant to killing by CART19 cells in a xenograft model yet retained sensitivity to anti-CD22 CAR T cells. In conclusion, our data therefore show that a single leukemic cell accidentally transduced with CAR19 survived the 10-day manufacturing process and, upon reinfusion into the patient, was the sole clone at relapse 9 months later. This leukemic clone evaded CTL019 detection via downregulation of the target antigen in a cell-autonomous fashion. Disclosures Lacey: Novartis: Research Funding. Xu:Novartis: Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Barrett:Novartis: Research Funding. Kulikovskaya:Novartis: Research Funding. Ambrose:Novartis: Research Funding. Patel:Novartis: Research Funding. Reich:Novartis: Research Funding. Scholler:Novartis: Patents & Royalties: Royalties, Research Funding. Nazimuddin:Novartis: Research Funding. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Maude:Novartis: Consultancy. Gill:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Patents & Royalties, Research Funding; GE Healthcare Bio-Sciences: Consultancy. Orlando:Novartis: Employment. Grupp:Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership; University of Pennsylvania: Patents & Royalties; Johnson & Johnson: Research Funding; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding. Melenhorst:Novartis: Patents & Royalties: Novartis, Research Funding.

Neurologic toxicity has been observed with anti-CD19 chimeric antigen receptor (CAR) T cells and the anti-CD19 BiTE blinatumomab. Both focal (e.g., cranial nerve palsy) and global (e.g., generalized seizures) abnormalities have been reported, often associated with systemic cytokine release syndrome (CRS) but also observed after recovery from or in absence of CRS. CART-BCMA consists of expanded autologous T cells transduced with a 4-1BB:CD3-zeta-based CAR specific for B Cell Maturation Antigen. Here, we report clinical features and management of a severe neurotoxicity observed on a phase 1 trial of CART-BCMA for multiple myeloma (MM) (NCT02546167). The subject is a 55-year-old female with high-risk IgA lambda MM. At time of CART-BCMA infusion, her MM manifestations included cytopenias and plasmacytomas of the pleura and paravertebral muscles. Bone marrow (BM) was >95% BCMA+ plasma cells. Pre-treatment brain MRI showed pachymeningeal thickening and enhancement over the left cerebral convexity, possibly due to extension of calvarial MM lesions. There was no evidence of CNS MM on a neurologist's exam or by CSF cytology. The subject received 2x108 CART-BCMA cells, 40% of the planned dose, over two consecutive days, without lymphodepleting chemotherapy; a third planned infusion was held due to fevers. Over the next 4 days, fevers persisted, hypoxia and delirium developed, and cytopenias worsened. Brain MRI and lumbar puncture on day 4 showed no new abnormalities. Evaluation for infection was negative. These symptoms coincided with rise in serum IL-6 (nl range On day 8, the subject became obtunded. She was intubated, and methylprednisolone 1000mg was administered followed by hydrocortisone 100mg q8h. By day 11, she was more alert, permitting extubation and cessation of steroids, but remained slow to respond. On day 12 she developed a generalized seizure, was re-intubated for airway protection, and received antiepileptic drugs (AED). She again improved and was extubated but, on day 15, developed status epilepticus that required five AEDs to control. Repeat MRI showed new diffuse sulcal, cortical, and subcortical T2/FLAIR signal abnormalities involving the bilateral frontal, parietal, occipital, posterior temporal, and cerebellar hemispheres, with sulcal effacement concerning for cerebral edema. This deterioration was not associated with fevers, other features of systemic CRS, or hypertension, but it coincided with rise in circulating CAR T cell frequency to one of the highest levels observed on our CAR trials (Figure, red line). CRS-related cytokines continued to decrease in serum, but CSF on day 16 showed marked elevation in IL-6, IL-8, and other CRS-related cytokines compared to day-16 serum and day-4 CSF (Figure, green line). CART-BCMA cells were detected in CSF. High-dose methylprednisolone was restarted without improvement. On day 17, cyclophosphamide 1.5 g/m2 was administered to reduce CART-BCMA cells. Beginning on day 18, mental status improved substantially, and she was extubated on day 20. Steroids were gradually tapered. MRI on day 23 showed near-resolution of signal abnormalities, which were completely resolved on MRI 4 weeks later. Neurologic signs and symptoms resolved; the last symptom to recover was mild difficulty processing visual information. Despite cyclophosphamide and a prolonged steroid course, CART-BCMA cells remained detectable in both blood and BM at last evaluation, 164 days after infusion, and the subject achieved a five-month VGPR. Conclusions: Given the rapid reversibility and MRI appearance, this neurologic syndrome was felt to be most consistent with posterior reversible encephalopathy syndrome (PRES), possibly due to high CSF levels of CRS-related cytokines, as opposed to encephalitis from direct CART-BCMA cytotoxicity against neuronal tissue. PRES developed amidst improvement of systemic CRS, suggesting a CNS-localized CRS, possibly due to occult CNS MM encountered by expanding CART-BCMA cells and/or failure of tocilizumab to block IL-6 receptor in the CNS. Cyclophosphamide may effectively reverse life-threatening, steroid-refractory CAR T cell neurotoxicity while retaining some CAR T cell efficacy and long-term persistence. Figure Figure. Disclosures Garfall: Medimmune: Consultancy; Bioinvent: Research Funding; Novartis: Consultancy, Research Funding. Lancaster:Grifols, Inc.: Other: Teaching courses; Medimmune, Inc.: Consultancy; Janssen: Consultancy. Stadtmauer:Teva: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Takada: Consultancy; Celgene: Consultancy. Lacey:Novartis: Research Funding. Ambrose:Novartis: Research Funding. Chen:Novartis: Research Funding. Kulikovskaya:Novartis: Research Funding. Vogl:Acetylon: Research Funding; GSK: Research Funding; Calithera: Research Funding; Teva: Consultancy; Celgene: Consultancy; Constellation: Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy. Plesa:Novartis: Patents & Royalties, Research Funding. Weiss:Novartis: Consultancy. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties: Novartis, Research Funding. Levine:Novartis: Patents & Royalties, Research Funding; GE Healthcare Bio-Sciences: Consultancy. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Tmunity Therapeutics: Equity Ownership; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Pfizer: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding. Cohen:Janssen: Consultancy; Bristol-Meyers Squibb: Consultancy, Research Funding.

Background : BCMA is expressed on MM cells, and CAR T cells targeting BCMA have pre-clinical anti-MM activity. CART-BCMA is an autologous T cell product engineered by lentiviral transduction to express a fully human BCMA-specific CAR with CD3ζ and 4-1BB signaling domains, and then expanded ex vivo using CD3/CD28 beads. Methods: In this ongoing, 3+3 dose-escalation study, relapsed/refractory MM patients (pts) receive CART-BCMA cells as split-dose infusions (10% on day 0, 30% on day 1, and 60% on day 2). Three cohorts are planned: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (CTX) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) CTX 1.5 g/m2 + 1-5 x 108 CART cells. Pts need serum creatinine (Cr) Results: To date, 11 pts have been screened, and 6 treated in cohort 1. Reasons for not receiving treatment were screen fail (n=2), rapid MM progression/renal failure (n=2), and pt/MD choice (n=1). The 6 treated pts were all IMID/PI-refractory with high risk cytogenetics and median 9 lines of therapy (Table). All expressed BCMA on MM cells, and achieved the minimum target dose of 1x108 CART-BCMA cells. All but 2 received 100% of planned dose, with 2 (pts 01and 03) receiving 40% (3rd infusions held for fever). Cytokine release syndrome (CRS) occurred in 5 patients: 2 grade 3 requiring tocilizumab (pts 01 and 03), 1 grade 2, and 2 grade 1. High-grade CRS was associated with elevated levels of IL-6, IFNg, MCP1, MIG, IL2Ra, and IL-10, as seen in our acute lymphoblastic leukemia CTL019 trial (Teachey et al, 2016). There was 1 DLT: grade 4 PRES (posterior reversible encephalopathy syndrome) in pt 03, with severe delirium, recurrent seizures, obtundation, and cerebral edema on MRI. This resolved after anti-epileptics, high-dose methylprednisolone and cyclophosphamide, without long-term neurologic sequelae. Other grade 3/4 toxicities to date include hypophosphatemia (n=3 pts), hypocalcemia (n=2), and anemia, neutropenia, lymphopenia, thrombocytopenia, hypofibrinogenemia, fatigue, pneumonia, UTI, elevated Alk phos and AST, hypokalemia, hypertension, and pleural effusion (n=1 each). CART-BCMA cells were detected in blood and marrow by CAR-specific PCR in all 6 pts, and in 4/6 by flow cytometry, with 2 pts, 01 and 03, having massive CART expansion peaking at 90% and 76% of peripheral CD3+ T cells, respectively. CART-BCMA cells during peak expansion were predominantly CD8+ and highly activated. Pt 01 has ongoing CART-BCMA persistence, with ongoing stringent CR at 7 months and MRD-negative bone marrow by flow cytometry. Pt 03, who had pleural and possible dural MM involvement, had CART-BCMA cells found in pleural fluid and CSF, and achieved VGPR (IF+ only) with resolution of extramedullary disease on PET/CT scan. She progressed at 5 months, associated with significant reduction of CART-BCMA cells and loss of BCMA expression on her MM cells by flow cytometry, suggestive of antigen escape. Two pts (02, 11) had modest CART-BCMA expansion, with 1 minimal response (MR) lasting 2 months, and 1 ongoing MR 1 month post-infusion. Two pts (09, 10) had minimal expansion and no response. Soluble BCMA levels, which were elevated in all pts at baseline, declined in parallel with CART-BCMA expansion and correlated with depth of response, with an accompanying increase in previously suppressed BAFF and APRIL levels in serum. Conclusions: CART-BCMA cells can be manufactured from heavily-pretreated MM pts, and demonstrate promising in vivo expansion and clinical activity, even without lymphodepleting conditioning. Depth of response correlates with degree of CART-BCMA expansion and CRS. Toxicities to date include CRS and in 1 pt, severe reversible neurotoxicity, as described in other CAR T cell studies. Expanded accrual in cohort 1, as well as in cohorts with CTX conditioning, is ongoing, with updated data to be presented at the meeting. Table Table. Disclosures Cohen: Bristol-Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy. Garfall:Bioinvent: Research Funding; Novartis: Consultancy, Research Funding; Medimmune: Consultancy. Stadtmauer:Novartis: Consultancy; Takada: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Teva: Consultancy; Janssen: Consultancy. Lacey:Novartis: Research Funding. Lancaster:Janssen: Consultancy; Medimmune, Inc.: Consultancy; Grifols, Inc.: Other: Teaching courses. Vogl:Millennium: Consultancy, Research Funding; Celgene: Consultancy; Karyopharm: Consultancy; Teva: Consultancy; Acetylon: Research Funding; Glaxo Smith Kline: Research Funding; Calithera: Research Funding; Constellation: Research Funding. Ambrose:Novartis: Research Funding. Plesa:Novartis: Patents & Royalties, Research Funding. Kulikovskaya:Novartis: Research Funding. Weiss:Prothena: Other: Travel, accommodations, Research Funding; Novartis: Consultancy; GlaxoSmithKline: Consultancy; Janssen: Consultancy, Other: Travel, accommodations, Research Funding; Millennium: Consultancy, Other: Travel, accommodations. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. Isaacs:Novartis: Employment. Melenhorst:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Tmunity: Equity Ownership, Other: Founder, stockholder ; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding.

Background: Though CD19 is expressed only rarely on multiple myeloma (MM) plasma cells (PC), rare CD19+ B cells can be identified in MM patients that are clonally related to the MM PC. These clonotypic B cells may exhibit properties of cancer stem cells (enhanced MM-propagating properties and drug resistance compared to MM PC) and thus be a potential therapeutic target in conjunction with therapies that target MM PC. CTL019 consists of autologous T cells transduced via lentiviral vector with an anti-CD19 scFv coupled to CD3-zeta and 4-1BB signaling domains and expanded ex vivo with anti-CD3/CD28-conjugated beads. To target both clonotypic B cells and MM PC, we conducted a pilot clinical trial of CTL019 administered after high-dose melphalan and autologous stem cell transplantation (ASCT) in relapsed/refractory MM patients who had previously undergone first-line ASCT with short progression-free survival (PFS). Methods: Subjects were required to be medically fit for ASCT and have progressed within 1 year of a prior ASCT performed as part of first-line therapy. Study therapy consisted of ASCT with melphalan 140-200 mg/m2 followed by 1-5x107 CTL019 cells 12-14 days later. The primary endpoint was safety and feasibility of CTL019 manufacturing and administration in this clinical setting. Secondary endpoints included assessments of CTL019 in vivo persistence and activity against normal B cells, plasma cell immunophenotype as a response biomarker, and PFS after ASCT + CTL019 in comparison to PFS after initial ASCT. Results: Twelve subjects enrolled, and 10 received study therapy; autologous T cells failed to expand ex vivo in one enrolled subject, and one enrolled subject elected to pursue off-study therapy. Median age was 61 (range 48-68). Median prior lines of therapy was 6 (range 2-10). Poor-prognosis features were present in 8/10 subjects (6/10 with poor-prognosis cytogenetics, 2/10 with BRAF V600E mutations, 1/10 with secondary plasma cell leukemia). Median PFS after first-line ASCT was 258 days (range 100-342). In pre-ASCT bone marrow (BM), the dominant MM PC population was CD19-negative by flow cytometry in 9/9 evaluable subjects, though 7/9 exhibited rare CD19+ subsets comprising 0.05-1.5% of MM PC. Melphalan dose was 140 (N=7) or 200 (N=3) mg/m2. All subjects infused received the maximum target dose of 5x107 CTL019 cells. Adverse events (AE) consisted mostly of expected ASCT toxicities. Grade ³3 AE that were at least possibly related to CTL019 included grade 3 autologous GVHD (N=1, resolved with corticosteroids) and oral mucositis (N=1). Grade 1 cytokine release syndrome occurred in 1 subject. There was no ASCT-related mortality. After infusion, CTL019 cells were detectable in peripheral blood (PB) of all subjects and persisted for median of 44 days (range 14-156). Presence of PB CTL019 cells was associated with absence of PB B cells. Notably, CTL019 cells were detected in BM in 9/10 subjects at day 42 and/or 100 post-ASCT. Median PFS after ASCT + CTL019 was 185 days (range 42-479); all subjects have progressed. The peak BM CTL019 frequency correlated significantly with favorable PFS (SpearmanÕs rho=0.77, P=0.009). There was no association between PFS and peak frequency of CTL019 or duration of CTL019 persistence in PB. In 3/10 subjects, PFS after ASCT + CTL019 met or exceeded PFS after first-line ASCT (Figure). For comparison, in a historical cohort of 18 patients who received first-line and salvage ASCT at our institution since 2008, no patients exhibited longer PFS after salvage ASCT. Conclusion: CTL019 manufacturing and administration post-ASCT is safe and feasible in patients with advanced MM. Correlation of PFS with CTL019 frequency in BM and prolonged PFS in 3 subjects is suggestive of clinical efficacy. A phase-two study of CTL019 using a 10-fold higher dose after first-line ASCT in high-risk MM patients is ongoing. Figure. Figure. Disclosures Garfall: Medimmune: Consultancy; Bioinvent: Research Funding; Novartis: Consultancy, Research Funding. Stadtmauer:Novartis: Consultancy; Takada: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Teva: Consultancy; Celgene: Consultancy. Maus:Novartis: Patents & Royalties: related to CTL019, Research Funding. Hwang:Novartis: Research Funding. Vogl:Takeda: Consultancy, Research Funding; GSK: Research Funding; Calithera: Research Funding; Teva: Consultancy; Constellation: Research Funding; Celgene: Consultancy; Acetylon: Research Funding; Karyopharm: Consultancy. Cohen:Bristol-Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy. Weiss:Novartis: Consultancy. Porter:Genentech: Employment; Novartis: Patents & Royalties, Research Funding. Frey:Novartis: Research Funding; Amgen: Consultancy. Milone:Novartis: Patents & Royalties, Research Funding. Mangan:Novartis: Speakers Bureau. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Patents & Royalties: Novartis, Research Funding. Ambrose:Novartis: Research Funding. Chen:Novartis: Research Funding. Kulikovskaya:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding; GE Healthcare Bio-Sciences: Consultancy. June:Johnson & Johnson: Honoraria; Tmunity Therapeutics: Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding; Pfizer: Honoraria.

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.

Background Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post autologous stem cell transplant (auto-SCT) may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies, in particular following post-transplant infusion of activated autologous T cells. However, efficacy of these approaches may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the low level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen-specific TCRs into autologous T cells infused post-transplant would overcome this limitation and improve response rates in the post auto-SCT setting. Methods We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an affinity-enhanced TCR that recognizes the NY-ESO-1/LAGE-1 peptide complex HLA‐A*0201‐SLLMWITQC; these cells are infused in the setting of profound lymphodepletion that accompanies high dose chemotherapy administered during auto-SCT. Patients with high risk or relapsed MM, who are HLA‐A*0201 positive, and whose tumor is positive for NY-ESO-1 and/or LAGE-1 by RT-PCR are eligible. CD25 depleted CD4 and CD8 T cells are activated and expanded using anti-CD3/CD28 antibody conjugated microbeads, and genetically modified with a lentiviral vector containing the TCR construct at a multiplicity of infection of 1. Engineered T cells are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1-10 billion total cells with a minimum gene modification requirement of 10%. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results Prior to enrollment on study, patients had received a median of 3 prior therapies including 6 with prior transplant. 50% of tumors contained high risk chromosomal abnormalities, and NY-ESO-1 expression is correlated with adverse prognosis. 20 patients (average age of 57) have been infused with an average of 2.3 X 109 engineered T cells (range 4.5 X 108-3.9 X 109); this reflects an average clinical scale transduction efficiency of 34% (range 18% – 49%). Infusions have been well tolerated, and the majority of adverse events were related to the high dose melphalan. Possibly related SAEs were neutropenia and thrombocytopenia, and GI and metabolism disorders including diarrhea, colitis, hyponatremia and hypomagnesemia. 10, 4, 2, and 2 patients have reached the 1 year, 9 month, 6 month and 3 month assessment timepoints, respectively, and 17/20 patients are alive. Best response by day 100 is sCR/CR in 2/15 (13%), nCR in 10/15 (67%), and PR in 3/15% (20%), which compares favorably to historic responses in patients undergoing first or second transplant. Engineered T cells expanded and persisted in blood and marrow at 180 days by Q-PCR and flow-cytometry in all but one case (Figure). 7 patients progressed after day 100, which was accompanied either by loss of engineered T cells or loss of tumor antigen. Detailed phenotyping and functional analysis of engineered T cells, and correlates with clinical responses, is underway. Summary This is the first clinical evaluation of engineered T cells in the MM setting. Infusions are safe, well tolerated, and are associated with encouraging responses in a high risk myeloma population. A study evaluating the engineered T cells in a non-transplant study is underway. Disclosures: Stadtmauer: Celgene: Consultancy. Binder-Scholl:Adaptimmune: Employment. Smethurst:Adaptimmune: Employment. Brewer:Adaptimmune: Employment. Bennett:Adaptimmune: Employment. Gerry:Adaptimmune: Employment. Pumphrey:Adaptimmune: Employment. Tayton-Martin:Adaptimmune: Employment. Ribeiro:Adaptimmune: Employment. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Jakobsen:Adaptimmune: Employment. Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. June:Novartis: Patents & Royalties, Research Funding.

Background Recent 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. Methods CAR 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. Results Detailed 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. Conclusions Adoptive 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.

Abstract 756 Background Advances in ex vivo T cell engineering have facilitated clinical trials to evaluate the potential for adoptive T cell transfer to target malignancy. Gene-modified T cells have the potential to expand, functionally persist and mediate long-term anti-tumor activity. Most clinical studies have shown only limited persistence of infused cells, with modest and often temporary anti-tumor activity. We reported initial clinical data on CAR T cells targeting CD19 expressed on normal and malignant B cells (CART19 cells) (Porter, et al NEJM 2011; Kalos et al Sci Trans Med 2011). The CAR included signaling domains from CD3zeta and CD137 (4-1BB) that mediated effector and proliferation/persistence signals. Here we report functional persistence of CART19 cells from the initial cohort at approximately 2 years post-infusion, and data from a more recently treated cohort of CLL patients and a pediatric patient with advanced, treatment refractory ALL. Methods Persistence of gene-modified T cells was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the CD137-TcR-zeta junction in the anti-CD19 CAR. CAR19 surface expression was detected by flow cytometry with an anti-CAR-19 idiotype specific antibody. B cell aplasia was evaluated by multiparametric flow cytometry. Multiplex cytokine analyses were performed using Luminex™assays. Results 10 patients with relapsed, refractory disease have been treated to date: 9 adults w/CLL and one child w/pre- B cell ALL. Each had been extensively pre-treated and had active disease at the time of CART19 infusion. All CLL patients received lymphodepleting chemotherapy 4–6 days before infusions, while the ALL patient did not get further lymphodepletion. Patients were infused with an average total of 1.7–50 × 108 total T cells which corresponded to 0.14–5.9 x108CART-19 cells. 9/10 treated patients were evaluable on 8.14.12. Detailed clinical outcomes will be reported separately at this meeting (Porter, D.L., Grupp S. et al). 4 pts (3 CLL, 1 ALL) had achieved CR at the primary endpoint (30 days post infusion) which is sustained and ongoing in all patients (range 1–24 months). Two CLL patients had a partial response (PR) lasting 3 and 5 months, while 3 patients did not respond (NR). In all patients with CR, robust in vivo expansion of CART19 cells was observed. By molecular analysis, CART19 cells demonstrated in vivo expansion, followed by contraction and an ongoing stable persistence at all evaluated timepoints. Expansion kinetics were unique for each patient; in all cases maximal expansion was observed by day +30 post CART-19 infusion. In patients with CR, observed peak marking for CART-19 ranged from 1 × 102-1 × 103 CART-19 cells/uL blood. Patients with PR demonstrated less robust in vivo expansion, with peak observed marking ∼1 × 101 CART-19 cells/uL blood. In NR patients, peak marking was Multiplex-cytokine analysis of serum samples from CR patients revealed a broad pro-inflammatory signature with significant elevation in a subset of soluble immune modulators including IL-6, IL-8, IFN-g, MIP1b, and IL2ra. In contrast, NR patients did not have elevated serum cytokines. In CR patients, elevation of cytokines tracked with expansion of CART19 cells and elimination of B cells, suggesting the potential for a cytokine-based diagnostic signature to monitor CART19 treatment efficacy. Conclusions Adoptive transfer of CART19 cells engineered to express CD137 and TCR-zeta signaling domains can result in in vivo expansion, homing to disease sites, and long-term functional persistence of CART19 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 ALL. A detailed cytokine profile and persistent B cell aplasia has been identified that may correlate with treatment efficacy. Disclosures: Kalos: University of Pennsylvania: Patents & Royalties. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

Abstract 755 Background: Adoptive transfer of T cells genetically engineered to express affinity-enhanced T cell receptors (TcRs) specific for self-antigens is one approach to generate potent anti-tumor immunity, and this approach is being evaluated in a number of clinical trials. Although persistence of engineered T cells is positively correlated with response rates, a recurring observation to-date has been poor T cell persistence beyond the initial treatment window. We have recently initiated a Phase II clinical trial (NCT01352286) in patients with recurrent multiple myeloma (MM), to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR that targets an epitope shared by the NY-ESO-1 and LAGE-1 cancer testis antigens commonly expressed by MM. Clinical study details and outcomes are reported elsewhere in this meeting (Rapoport, A, Stadtmauer, E. et al.). Here we report the in vivo expansion, bioactivity, trafficking, durable persistence, and anti-tumor activity of TcR modified cells in this trial. Methods: Subjects received high-dose melphalan, infusion of autologous hematopoietic stem cells on day 0, and infusion of gene engineered cells (GEC) on day +2. Persistence of GEC was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the lentivirus backbone sequence. Surface expression of the introduced affinity-enhanced TcR α/β receptor was detected by multiparametric flow cytometry and TcR-specific dextramer™reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow samples was assessed by quantitative ABI-taqman based PCR and inventoried primer/probe sets. Results: As of August 2012, we have screened marrow from 46 patients for NY-ESO- and LAGE-1 expression. Of these, 29 (63%) and 17 (37%) expressed LAGE-1 and NY-ESO-1, respectively by PCR. LAGE-1 co-expression was observed in all cases with NY-ESO-1. To date 12 patients have been infused with GEC. Robust engraftment was observed, with peak levels of gene-marked cells in peripheral blood and marrow ranging from 1 × 102-1 × 103 cells/μL and peak levels detected within the first 21 days post infusion. By day +100 the absolute number of GEC showed patient-specific and variable contraction, with a subset of patients maintaining GEC at 1 × 102-1 × 103 cells/μL, and a second subset where GEC contracted to 1 × 101-1 × 102cells/μL. Trafficking to and persistence of GEC in marrow was observed. At both early (up to day +30) and, in a subset of patients, late timepoints (post day+42), circulating CD3+ T cells could be detected with surface expression of the affinity enhanced TcR. Engineered cells are detected in all patients at their latest timepoint by Q-PCR. To date we see no evidence for TCR mispairing with endogenous TCR. Tumor-antigen and tumor-specific targeting by GEC was evaluated using Q-PCR and quantifying transcript levels of NY-ESO-1, LAGE-1, and CD138 (as a plasma cell marker) in marrow biopsies at enrollment, and days +42, +100, and +180. In the initial cohort of 5 patients, selective reappearance of CD138 transcript is observed in 4 cases (2 sCR, 1 VGPR, 1 PR), with non-detectable or very weak expression of LAGE-1/NY-ESO-1 transcripts, suggesting specific targeting of tumor cells that express LAGE-1/NY-ESO-1. In the fifth patient, who has progressive disease and low engineered cell persistence, CD138 and LAGE-1/NY-ESO-1 transcripts are both present above pre-treatment levels. A second infusion was recently given to this patient. Summary: Our studies show for the first time that adoptive transfer of affinity-enhanced TCR engineered T cells in MM patients results in prolonged T cell persistence, homing to marrow, and anti-tumor activity. Disclosures: Binder-Scholl: Adaptimmune: Employment. Smethurst:Adaptimmune Ltd: Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.