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CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has become a breakthrough treatment of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, despite the high initial response rate, the majority of adult patients with B-ALL progress after CD19 CAR T-cell therapy. Data on the natural history, management, and outcome of adult B-ALL progressing after CD19 CAR T cells have not been described in detail. Herein, we report comprehensive data of 38 adult patients with B-ALL who progressed after CD19 CAR T therapy at our institution. The median time to progression after CAR T-cell therapy was 5.5 months. Median survival after post–CAR T progression was 7.5 months. A high disease burden at the time of CAR T-cell infusion was significantly associated with risk of post–CAR T progression. Thirty patients (79%) received salvage treatment of post–CAR T disease progression, and 13 patients (43%) achieved complete remission (CR), but remission duration was short. Notably, 7 (58.3%) of 12 patients achieved CR after blinatumomab and/or inotuzumab administered following post–CAR T failure. Multivariate analysis revealed that a longer remission duration from CAR T cells was associated with superior survival after progression following CAR T-cell therapy. In summary, overall prognosis of adult B-ALL patients progressing after CD19 CAR T cells was poor, although a subset of patients achieved sustained remissions to salvage treatments, including blinatumomab, inotuzumab, and reinfusion of CAR T cells. Novel therapeutic strategies are needed to reduce risk of progression after CAR T-cell therapy and improve outcomes of these patients.

Chimeric antigen receptor (CAR) T cells have demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We undertook a multicenter clinical trial to determine toxicity, feasibility, and response for this therapy. A total of 25 pediatric/young adult patients (age, 1-22.5 years) with R/R B-ALL were treated with 19-28z CAR T cells. Conditioning chemotherapy included high-dose (3 g/m2) cyclophosphamide (HD-Cy) for 17 patients and low-dose (≤1.5 g/m2) cyclophosphamide (LD-Cy) for 8 patients. Fifteen patients had pretreatment minimal residual disease (MRD

Factors contributing to hematopoietic recovery following chimeric antigen receptor (CAR) T-cell therapy have not been well studied. In an analysis of 83 patients with hematologic malignancies treated with CAR T-cell therapy, we describe patterns of hematopoietic recovery and evaluate potentially associated factors. We included patients who received axicabtagene ciloleucel (n = 30) or tisagenlecleucel (n = 10) for B-cell lymphoma, CD19-28z CAR T therapy for B-cell acute lymphoblastic leukemia (NCT01044069; n = 37), or B-cell maturation antigen targeting CAR T cells for multiple myeloma (NCT03070327; n = 6). Patients treated with CAR T cells who had not progressed, died, or received additional chemotherapy had “recovered” (per definition in Materials and methods section) hemoglobin, platelet, neutrophil, and white blood cell counts at rates of 61%, 51%, 33%, and 28% at month 1 postinfusion and 93%, 90%, 80%, and 59% at month 3 postinfusion, respectively. Univariate analysis showed that increasing grade of immune effector cell–associated neurological syndrome (ICANS), baseline cytopenias, CAR construct, and higher peak C-reactive protein or ferritin levels were statistically significantly associated with a lower likelihood of complete count recovery at 1 month; a similar trend was seen for cytokine release syndrome (CRS). After adjustment for baseline cytopenia and CAR construct, grade ≥3 CRS or ICANS remained significantly associated with the absence of complete count recovery at 1 month. Higher levels of vascular endothelial growth factor and macrophage-derived chemokines, although not statistically significant, were seen patients without complete count recovery at 1 month. This remains to be studied further in larger prospective studies.

Background: There is compelling rationale to use bridging radiotherapy (BRT) between leukapheresis and chimeric antigen receptor T-cell infusion for cytoreduction and palliation of relapsed/refractory B cell malignancies. There is also preclinical evidence suggesting possible immune synergy with the combination of BRT and CAR T. Existing BRT series are small and generally focused on diffuse large B-cell lymphoma (DLBCL). We report a large cohort of RT-bridged patients including early experience with BRT for non-DLBCL histologies. Methods: We analyzed 43 patients with DLBCL (n=35), mantle cell lymphoma (MCL; n=6), and Burkitt's lymphoma (BL; n=2). All received RT for any intent within 1 month prior to leukapheresis up until CAR T infusion. PET response was evaluated by Lugano criteria within the BRT treatment field ("in field"), outside of the BRT field ("out of field") and overall at 5 time points: pre-BRT, pre-CAR T infusion, and day +30 (n=41), +90 (n=30), and +180 (n=19). Survival analysis was per Kaplan-Meier and clincodemographic associations with survival were assessed by Cox univariate proportional hazards. Results: Patients received axicabtagene (n=20, 48%), tisagenlecleucel (n=11, 26%), lisocabtagene (n=8, 19%), brexucabtagene (n=2, 5%), and an experimental CAR T product (n=1, 2%). Overall, patients had a median of 3 prior therapies (range 1-9). Prior to BRT, most (74%) had advanced stage III/IV relapsed/refractory disease and 17 (40%) had bulky (>7.5 cm) lesions. There was heterogeneity in BRT-treatment parameters; most commonly treated sites were head/neck (n=10), pelvis/groin (n=8), and abdomen (n=7). Median BRT dose was 30 Gy (range 4-54), with 20 Gy in 5 fractions as the most common regimen (n=12). 18 (42%) received BRT to all sites of PET avid disease ("comprehensive BRT"). 16 (37%) patients also received systemic bridging therapy, including concurrently (n=10) with BRT. With a short 14d median time to restaging PET, in field overall response rate (ORR) post BRT was 82% (complete response, CR; n=9, 27% or partial response, PR; n=18, 55%) [Figure 1]. Reflective of the population referred for bridging, 21 (64%) had out of field progression post BRT and pre-CAR T. Post-CAR T, 2 patients (5%) had grade 3 cytokine release syndrome, 4 (9%) had grade 3 neurotoxicity, and no severe toxicity within the BRT field was noted. Following CAR T, ORR was 72% (CR: n=25, 60%; PR: n=5, 12%). Median post CAR T follow-up was 9 mos (range 1-40), with 44% (n=19) of patients having no evidence of disease, 44% (n=19) deceased, and 12% (n=5) alive with disease at last contact. 33 (77%) patients never progressed within the BRT field. Of the 27 (63%) patients who relapsed, 10 were CD19- and 9 (33%) had disease in the BRT field at first progression. In DLBCL patients, progression free survival (PFS) was 83%, 60%, and 51% and overall survival (OS) was 100%, 89% and 69%, respectively at Day +30, +90, and +180. Univariate analysis showed that pre-BRT ECOG performance status ≥2 (HR: 12.0, p Conclusion: BRT has broad utility for aggressive B-cell malignancies and is associated with excellent pre-CAR T local control and no serious toxicity within the irradiated sites. Most patients had durable post-CAR T local control. Prospective studies are planned to clarify outcomes and evaluate mechanistic synergies. Future studies are needed to define optimal patient characteristics for systemic vs. radiotherapy bridging and elucidate the best BRT strategy for maximizing local control and potential immune augmentation. Figure 1 Figure 1. Disclosures Palomba: Kite: Consultancy; Novartis: Consultancy; Notch: Honoraria, Other: Stock; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Lygenesis: Honoraria; PCYC: Consultancy; BeiGene: Consultancy; Pluto: Honoraria; Wolters Kluwer: Patents & Royalties; Juno: Patents & Royalties; Ceramedix: Honoraria; Magenta: Honoraria; Priothera: Honoraria; Nektar: Honoraria; WindMIL: Honoraria; Rheos: Honoraria. Shouval: Medexus: Consultancy. Batlevi: Regeneron: Current holder of individual stocks in a privately-held company; Viatris: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; Pfizer: Current holder of individual stocks in a privately-held company; Medscape: Honoraria; TG Therapeutics: Consultancy; Seattle Genetics: Consultancy; Life Sciences: Consultancy; Kite Pharma: Consultancy; Dava Oncology: Honoraria; Karyopharm: Consultancy; Juno/Celgene: Consultancy; Bayer: Research Funding; Memorial Sloan Kettering Cancer Center: Current Employment; ADC Therapeutics: Consultancy; GLG Pharma: Consultancy; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Brentjens: BMS: Consultancy, Patents & Royalties, Research Funding; Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; sanofi: Patents & Royalties; Caribou: Patents & Royalties. Dahi: Kite / Gilead: Membership on an entity's Board of Directors or advisory committees. Giralt: JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Park: Kura Oncology: Consultancy; Intellia: Consultancy; Autolus: Consultancy; Artiva: Consultancy; Novartis: Consultancy; Amgen: Consultancy; PrecisionBio: Consultancy; Curocel: Consultancy; Affyimmune: Consultancy; BMS: Consultancy; Minerva: Consultancy; Servier: Consultancy; Kite Pharma: Consultancy; Innate Pharma: Consultancy. Scordo: i3 Health: Other: Speaker; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; Omeros Corporation: Consultancy; Angiocrine Bioscience: Consultancy, Research Funding; McKinsey & Company: Consultancy. Sauter: Gamida Cell: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Genmab: Consultancy; GSK: Consultancy; Bristol-Myers Squibb: Research Funding; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Shah: Janssen: Research Funding; Amgen: Research Funding. Perales: NexImmune: Honoraria; Kite/Gilead: Honoraria, Other; Karyopharm: Honoraria; Bristol-Myers Squibb: Honoraria; Cidara: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Celgene: Honoraria; MorphoSys: Honoraria; Medigene: Honoraria; Omeros: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; Miltenyi Biotec: Honoraria, Other; Merck: Honoraria; Sellas Life Sciences: Honoraria; Servier: Honoraria; Takeda: Honoraria.

Background: Patients with diffuse large B cell lymphoma (DLBCL) who do not achieve a complete response (CR) following CD19-targeted Chimeric Antigen Receptor T cell (CAR T) therapy are less likely to obtain durable benefit. Greater tumor burden pre-CAR T has been shown to predict lower CR rate and shorter survival. Recently, patterns of failure studies have identified baseline lesion characteristics including size, SUV, and extranodal location as associated with increased risk of post-CAR T failure. It is not yet known how bridging radiotherapy (BRT) prior to CAR T can alter subsequent patterns of failure. Methods: We reviewed DLBCL patients treated from 2017 to 2021 with BRT for any intent in the period from 30d pre-leukapheresis to CAR T infusion. Comprehensive BRT fields were defined as no avidity above liver mean outside of the BRT field. Bulky disease was defined as a max diameter of ≥7.5cm in any dimension. PET response was evaluated by Lugano criteria. Pattern of failure analysis was performed to identify failure sites as pre-existing (present pre-CAR T) vs. new and as in-field, marginal, or distant with respect to BRT. Marginal was defined as failure outside of the area receiving prescription dose but within 1cm of BRT field edge. One patient with a cutaneous target was excluded from size-based analyses. Significance was assessed by Fisher's exact test or T-test. Results: Thirty-five patients were identified with a median age of 66. Most were advanced stage (74%) at BRT with a median of 3 prior lines of systemic therapy. Twelve (35%) had bulky disease at any site pre-BRT (median max diameter 5.9 cm; range 1.1 - 24) with highest overall SUV median 19.8 (range 3.4 - 47); 83% (n=29) had ≥1 extranodal site (bone: n=13; CNS: n=4). Most common BRT sites were head/neck (n=10), pelvis (n=6), and extremity (n=5). BRT targeted the largest lesion in 85% (median 5.8 cm; 32% bulky) and the site of highest SUV in 81% (median 18.7); 49% (n=17) of targets were extranodal. BRT fields were comprehensive in 39%; median radiation treatment volume was 751cc (range 18 - 5856). Twenty-four patients (63%) received ≥30 Gy (median 30 Gy; range 20 - 54). Systemic therapy was given during the bridging period to 31% (n=11). Patients received axicabtagene (n=20), tisagenlecleucel (n=11), lisocabtagene (n=3), or experimental CAR T (n=1). On PET after BRT and pre-infusion (median 12d from BRT), 85% (n=23) had achieved partial response (PR) or better in-field (CR: n=7) though 63% had kinetically active disease with out-of-field progression (PD) and 19% (n=5) had marginal PD. Despite short interval restaging, there was a significant reduction in patients with bulky disease after BRT both within the irradiated field (p=0.007) and at any site (p=0.01). (Fig 1a) A median 81% SUV reduction in the irradiated site was noted with a significant decrease in max SUV in both the irradiated (p Conclusion: BRT significantly reduced the number of patients with bulky disease at any site at time of CAR T and significantly reduced the overall highest lesional SUV. Marginal failure rates of 19% and 23% at interim scan post-BRT and after CAR T, respectively in conjunction with the pattern of post CAR T failure predominantly in pre-existing sites, may suggest a need for more generous fields when a BRT approach is used in this population with rapidly proliferating disease. Further work and larger sample sizes are needed to evaluate the impact of dose on local control in bulky lesions as well as to investigate whether comprehensive BRT affects outcomes or patterns of failure. Figure 1 Figure 1. Disclosures Palomba: Juno: Patents & Royalties; Notch: Honoraria, Other: Stock; Novartis: Consultancy; Kite: Consultancy; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Magenta: Honoraria; Ceramedix: Honoraria; WindMIL: Honoraria; Nektar: Honoraria; Rheos: Honoraria; Priothera: Honoraria; BeiGene: Consultancy; Lygenesis: Honoraria; Pluto: Honoraria. Shouval: Medexus: Consultancy. Batlevi: Kite Pharma: Consultancy; Viatris: Current holder of individual stocks in a privately-held company; Dava Oncology: Honoraria; TG Therapeutics: Consultancy; ADC Therapeutics: Consultancy; Regeneron: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; Juno/Celgene: Consultancy; Medscape: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Seattle Genetics: Consultancy; Life Sciences: Consultancy; Pfizer: Current holder of individual stocks in a privately-held company; Karyopharm: Consultancy; Moderna: Current holder of individual stocks in a privately-held company; Memorial Sloan Kettering Cancer Center: Current Employment; Bayer: Research Funding; GLG Pharma: Consultancy; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Brentjens: Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; BMS: Consultancy, Patents & Royalties, Research Funding; sanofi: Patents & Royalties; Caribou: Patents & Royalties. Dahi: Kite / Gilead: Membership on an entity's Board of Directors or advisory committees. Giralt: JAZZ: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Park: BMS: Consultancy; Kite Pharma: Consultancy; Innate Pharma: Consultancy; Novartis: Consultancy; Minerva: Consultancy; Servier: Consultancy; Kura Oncology: Consultancy; PrecisionBio: Consultancy; Amgen: Consultancy; Intellia: Consultancy; Artiva: Consultancy; Curocel: Consultancy; Autolus: Consultancy; Affyimmune: Consultancy. Scordo: Angiocrine Bioscience: Consultancy, Research Funding; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; i3 Health: Other: Speaker; Omeros Corporation: Consultancy; McKinsey & Company: Consultancy. Sauter: Bristol-Myers Squibb: Research Funding; GSK: Consultancy; Gamida Cell: Consultancy; Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Celgene: Consultancy, Research Funding; Genmab: Consultancy; Novartis: Consultancy; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Shah: Amgen: Research Funding; Janssen Pharmaceutica: Research Funding. Perales: Merck: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Cidara: Honoraria; Miltenyi Biotec: Honoraria, Other; Kite/Gilead: Honoraria, Other; Equilium: Honoraria; Incyte: Honoraria, Other; Medigene: Honoraria; Karyopharm: Honoraria; Takeda: Honoraria; Servier: Honoraria; NexImmune: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; Sellas Life Sciences: Honoraria; Omeros: Honoraria; MorphoSys: Honoraria.

Background: CD19 CAR T cells have demonstrated high response rates in patients (pts) with relapsed or refractory (R/R) lymphoma, but these therapies are associated with high rates of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), often requiring prolonged hospital stay including transfer to the intensive care unit (ICU). Real-world data and early and prophylactic corticosteroid trials with axicabtagene in R/R large cell lymphoma have reported lower rates of severe CRS to Methods: Adult pts with R/R large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL) receiving commercially available CD19 CAR T cells were enrolled. Pts received anakinra 100mg subcutaneous every 12 hours starting either on day 2 of CAR T cell infusion or after 2 documented fevers of ≥38.5 prior to day 2, whichever was earlier. Anakinra was continued for a minimum of 10 days, and the dose could be increased to 100mg every 6 hours and continued beyond 10 days in the case of persistent or progressing CRS and ICANS. Pts received tocilizumab and/or corticosteroids after anakinra initiation for persistent or worsening CRS or ICANS. CRS and ICANS were assessed per the ASTCT consensus grading. Disease response was assessed per the Lugano criteria. The primary objective was to determine the rate of severe ICANS within the first 28 days of CAR T cell infusion. Secondary objectives included assessment of severe CRS, all grades of CRS and ICANS, disease response and serum and CSF cytokines. Results: A total of 31 pts (LBCL=27, MCL=4) were enrolled to the study. The median age of the pts at the time of T cell infusion was 62 (range, 30-77). CD19 CAR products included axicabtagene (23 pts; 74%), tisagenlecleucel (4 pts; 13%) and brexucabtagene (4 pts; 13%). 17 pts (55%) received bridging therapy prior to T cell infusion. All pts started anakinra at 100mg q12h; 25 pts started on day 2 and 6 pts prior to day 2 for grade 1 CRS. Anakinra dose was increased to 100mg q6h in 13 pts (42%). The median duration of anakinra administration was 10 days (range, 10-27). CRS of all grades was observed in 21 pts (68%) with severe CRS (grade 3-4) in 2 pts (6%) (1 with axicabtagene and 1 with brexucabtagene) (Table). Median CRS duration was 5.5 days (range, 1-21). ICANS of all grades was observed in 4 pts (13%), with severe ICANS in 2 pts (6%) (both grade 3; 1 with axicabtagene and 1 with brexucabtagene). No pt experienced grade 5 CRS or ICANS. Tocilizumab and corticosteroids were used in 9 pts (29%) and 6 pts (19%), respectively. Three pts (10%) required ICU transfer (2 with brexucabtagene and 1 with axicabtagene). In LBCL pts treated with axicabtagene (n=23), both severe CRS and ICANS were observed in 4% with tocilizumab and steroid use in 22% and 13%, respectively (Table). With a median follow-up of 104 days (range, 21-363), overall disease response rate at month 1 was 74% for all pts, with CR rate at 1 and 3 months of 58% and 52%, respectively. Conclusion: Early use of IL-1 receptor inhibitor anakinra appears to be safe and feasible, and reduces the rates of both severe CRS and ICANS with the comparable response rates in adult pts with R/R B-cell lymphoma receiving CD19 CAR T cells. The overall severe CRS and ICANS rates were 6% each with relatively low utilization of tocilizumab (29%) and corticosteroids (19%). In pts receiving axicabtagene, the rate of severe ICANS was 4%. A longer follow-up is needed to assess durability of remission, but the study provides strong support for continued investigation of IL-1 inhibition in prevention of severe ICANS. Further exploratory analysis is planned to address the impact of anakinra on changes in serum and CSF cytokines. Figure 1 Figure 1. Disclosures Park: BMS: Consultancy; Servier: Consultancy; Minerva: Consultancy; Curocel: Consultancy; Autolus: Consultancy; PrecisionBio: Consultancy; Intellia: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy; Artiva: Consultancy; Kura Oncology: Consultancy; Novartis: Consultancy; Affyimmune: Consultancy; Innate Pharma: Consultancy. Sauter: Gamida Cell: Consultancy; GSK: Consultancy; Bristol-Myers Squibb: Research Funding; Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Celgene: Consultancy, Research Funding; Genmab: Consultancy; Novartis: Consultancy; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Palomba: Juno: Patents & Royalties; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Notch: Honoraria, Other: Stock; Novartis: Consultancy; Kite: Consultancy; Wolters Kluwer: Patents & Royalties; PCYC: Consultancy; Ceramedix: Honoraria; Lygenesis: Honoraria; Magenta: Honoraria; BeiGene: Consultancy; WindMIL: Honoraria; Rheos: Honoraria; Nektar: Honoraria; Priothera: Honoraria; Pluto: Honoraria. Shah: Amgen: Research Funding; Janssen Pharmaceutica: Research Funding. Dahi: Gilead sciences: Membership on an entity's Board of Directors or advisory committees; Kite pharma: Membership on an entity's Board of Directors or advisory committees. Scordo: Angiocrine Bioscience: Consultancy, Research Funding; Omeros Corporation: Consultancy; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; i3 Health: Other: Speaker; McKinsey & Company: Consultancy. Batlevi: Kite Pharma: Consultancy; TouchIME: Honoraria; Seattle Genetics: Consultancy; Juno/Celgene: Consultancy; Karyopharm: Consultancy; TG Therapeutics: Consultancy; ADC Therapeutics: Consultancy; Medscape: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; Regeneron: Current holder of individual stocks in a privately-held company; Viatris: Current holder of individual stocks in a privately-held company; Pfizer: Current holder of individual stocks in a privately-held company; Dava Oncology: Honoraria; Life Sciences: Consultancy; Memorial Sloan Kettering Cancer Center: Current Employment; Bayer: Research Funding; GLG Pharma: Consultancy; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Perales: Merck: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Equilium: Honoraria; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other; MorphoSys: Honoraria; Takeda: Honoraria; Medigene: Honoraria; Incyte: Honoraria, Other; Cidara: Honoraria; Karyopharm: Honoraria; Kite/Gilead: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; Sellas Life Sciences: Honoraria; NexImmune: Honoraria; Omeros: Honoraria; Novartis: Honoraria, Other. Santomasso: Janssen: Consultancy; Legend: Consultancy; Kite/Gilead: Consultancy; Celgene/BMS: Consultancy; Incyte: Consultancy; In8bio: Consultancy. Sadelain: Juno Therapeutics: Patents & Royalties; NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); Minerva Biotechnologies: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; St. Jude Children's Research Hospital: Other: Provision of Services; Ceramedix: Patents & Royalties; Atara Biotherapeutics: Patents & Royalties. Brentjens: BMS: Consultancy, Patents & Royalties, Research Funding; Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; sanofi: Patents & Royalties; Caribou: Patents & Royalties. OffLabel Disclosure: Anakinra for prevention of CRS and ICANS associated with CD19 CAR

Introduction: Cellular immunotherapy with CD19-targeted chimeric antigen receptor (CAR) T cells has provided new therapeutic options for patients with high-risk hematologic malignancies. Following this therapy, patients may experience disease relapse or CAR-mediated toxicity due to cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Recent studies have confirmed that the intestinal microbiome can modulate the anti-tumor immune response to chemotherapy, immune checkpoint blockade, graft-versus-host disease after allogeneic hematopoietic cell transplantation, and adoptive cellular therapy. The contribution of the intestinal microbiome on the function of CAR T cells in vivo both with respect to their anti-tumor function and their propensity to induce toxicities is not known. Hence, in a multi-center study we analyzed the association between clinical outcomes and (1) antibiotic exposure prior to CAR T cell infusion and (2) the composition and diversity of the fecal microbiome. Methods and Results: We retrospectively collected clinical data and antibiotic exposures from patients with acute lymphoblastic leukemia (ALL, n=91) and non-Hodgkin lymphoma (NHL, n=137) treated with investigational or commercial CD19 CAR T cells at Memorial Sloan Kettering Cancer Center (MSK) and the University of Pennsylvania (Penn). We considered any antibiotic exposure between day -30 and the day of CAR T cell infusion. We focused our analysis on anaerobe-targeting antibiotics used in the setting of neutropenic fever: piperacillin-tazobactam, imipenem-cilastatin, and meropenem (here referred to as "P-I-M"). We found that forty-seven (20.6%) of 228 patients were exposed to P-I-M in the four weeks before CAR T cell infusion. Patient characteristics at the time of CAR T cell infusion were similar between the P-I-M-exposed and not-exposed groups, although a worse performance status was observed in patients with NHL treated with P-I-M. We found that overall survival (OS) was significantly decreased following CAR T cell infusion in patients exposed to P-I-M (Fig 1A; OS HR, 2.58; 95% CI, 1.68 - 3.98; p= We also prospectively collected baseline fecal samples prior to cell infusion from CD19 CAR T cells recipients (n=48) at MSK and Penn. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the amplicon sequence variants (ASVs) were annotated according to the NCBI 16S database using BLAST. In comparison to healthy controls (n=30), we found that alpha-diversity was significantly lower in fecal samples from CAR T cell patients (p= 0.0023, Fig 1B) and the composition of fecal samples was significantly different (p= Conclusion: Our results suggest that exposure to antibiotics, in particular P-I-M, in the four weeks before therapy was associated with worse survival. Profiling of the baseline fecal microbiome samples by 16S revealed that CD19 CAR T cell patients presented with evidence of an altered fecal microbiome as measured by lower alpha-diversity and a composition that is distinct from that of healthy controls. Finally, we identified bacterial taxa that were associated with Day 100 CR and CAR-mediated toxicity. Our findings indicate that the intestinal microbiome can affect the efficacy of CD19 CAR T cell therapy and provides a rationale to target the intestinal microbiome to improve clinical outcomes of patients treated with cellular therapies. Figure 1 Figure 1. Disclosures Smith: Janssen: Consultancy, Honoraria. Gomes: Xbiome: Current Employment. Schluter: Postbiotics Plus LLC: Other: cofounder. Park: Kura Oncology: Consultancy; BMS: Consultancy; Servier: Consultancy; Autolus: Consultancy; Curocel: Consultancy; Artiva: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Affyimmune: Consultancy; Intellia: Consultancy; Innate Pharma: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy. Palomba: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Jain: Targeted Healthcare Communications: Consultancy; Bristol Myers Squibb: Other: for advisory board participation; CareDx: Other: for advisory board participation; CTI Biopharma: Research Funding; Syneos Health: Research Funding. Pennisi: Gilead Sciences: Consultancy. Perales: Miltenyi Biotec: Honoraria, Other; Novartis: Honoraria, Other; Omeros: Honoraria; NexImmune: Honoraria; Bristol-Myers Squibb: Honoraria; Merck: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Nektar Therapeutics: Honoraria, Other; Cidara: Honoraria; Servier: Honoraria; Sellas Life Sciences: Honoraria; Karyopharm: Honoraria; MorphoSys: Honoraria; Equilium: Honoraria; Incyte: Honoraria, Other. Garfall: Amgen: Honoraria; CRISPR Therapeutics: Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Novartis: Research Funding; Tmunity: Research Funding. Landsburg: Triphase: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: DSMB member; Incyte: Membership on an entity's Board of Directors or advisory committees; ADCT: Membership on an entity's Board of Directors or advisory committees; Curis: Research Funding; Takeda: Research Funding. Gerson: Kite: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy; TG Therapeutics: Consultancy. Svoboda: Imbrium: Consultancy; Genmab: Consultancy; Astra Zeneca: Consultancy, Research Funding; Atara: Consultancy; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Incyte: Research Funding; Merck: Research Funding; Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; TG: Research Funding. Giralt: AMGEN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding. Rivière: FloDesign Sonics: Other: Provision of Services; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); Juno Therapeutics: Patents & Royalties. Porter: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Wiley and Sons Publishing: Honoraria; Tmunity: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; ASH: Membership on an entity's Board of Directors or advisory committees; DeCart: Membership on an entity's Board of Directors or advisory committees; Genentech: Current equity holder in publicly-traded company, Ended employment in the past 24 months; American Society for Transplantation and Cellular Therapy: Honoraria; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schuster: Abbvie: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Adaptive Biotechnologies: Research Funding; BeiGene: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; DTRM: Research Funding; Genetech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Incyte: Research Funding; Juno Theraputics: Consultancy, Research Funding; Loxo Oncology: Consultancy; Merck: Research Funding; Nordic Nanovector: Consultancy; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; Pharmaclcyclics: Research Funding; Tessa Theraputics: Consultancy; TG Theraputics: Research Funding. Sadelain: NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Atara Biotherapeutics: Patents & Royalties; Ceramedix: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; St. Jude Children's Research Hospital: Other: Provision of Services; Juno Therapeutics: Patents & Royalties; Minerva Biotechnologies: Patents & Royalties. Frey: Novartis: Research Funding; Kite Pharma: Consultancy; Sana Biotechnology: Consultancy; Syndax Pharmaceuticals: Consultancy. Brentjens: Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; BMS: Consultancy, Patents & Royalties, Research Funding; sanofi: Patents & Royalties; Caribou: Patents & Royalties. June: AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy; Novartis: Patents & Royalties; Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company. Pamer: Diversigen: Other: Advisory board; Bristol Myers Squibb, Celgene, Seres Therapeutics, MedImmune, Novartis and Ferring Pharmaceuticals: Honoraria. Peled: DaVolterra: Consultancy; MaaT Pharma: Consultancy; CSL Behring: Consultancy; Seres Therapeutics: Research Funding. Ruella: BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties; viTToria biotherapeutics: Research Funding. van den Brink: WindMILTherapeutics: Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; Priothera: Research Funding; Forty-Seven, Inc.: Honoraria; MagentaTherapeutics: Honoraria; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Merck & Co, Inc: Honoraria; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Kite Pharmaceuticals: Other; Amgen: Honoraria; Frazier Healthcare Partners: Honoraria; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options; Rheos: Honoraria; Therakos: Honoraria; Jazz Pharmaceuticals: Honoraria; Notch Therapeutics: Honoraria; Nektar Therapeutics: Honoraria; Wolters Kluwer: Patents & Royalties; Juno Therapeutics: Other; DKMS (nonprofit): Other; Pharmacyclics: Other; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards .

Background: BCMA targeted CAR T cell therapy has shown promising results in patients with relapsed/refractory multiple myeloma (RRMM), but relapses are common. Additional treatment options with novel therapeutic targets or mechanisms of action are needed. Here we report on the safety and efficacy of MCARH109, the first-in-class G Protein Coupled Receptor Class C Group 5 Member D (GPRC5D) targeted CAR T cell therapy (Smith EL et al. Sci. Trans Med 2019) in RRMM including patients who relapsed post BCMA targeted CAR T cell therapy. Methods: This is a phase I first-in-human, dose escalation trial of MCARH109; patients received lymphodepleting chemotherapy with fludarabine 30 mg/m 2 daily and cyclophosphamide 300 mg/m 2 daily for 3 days followed by a single infusion of MCARH109. The trial followed a standard 3+3 design with the following dose cohorts to date: 25X10 6, 50X10 6, 150X10 6, 450X10 6 viable CAR + T cells. The primary objective is to assess safety of MCARH109; secondary objectives include anti-myeloma efficacy, expansion and persistence of MCARH109 using quantitative polymerase chain reaction (qPCR) on peripheral blood and bone marrow samples. Results: 18 patients with RRMM were enrolled and underwent apheresis between September 15, 2020 and July 14, 2021. 12 patients have completed MCARH109 infusion to date, with 6 patients currently undergoing manufacturing and pending treatment. Of the 12 patients treated, median age was 59 (37-76) years and patients received a median of 8 (4-14) lines of therapy. 11 (92%) were penta-exposed, all patients were triple refractory, and 7 (58%) had prior treatment with BCMA targeted therapy including 6 (50%) who received prior BCMA CAR T therapy. 3 (25%) patients had non-secretory myeloma and 6(50%) patients had extramedullary plasmacytoma at baseline. 11 (92%) were refractory to last line of therapy and 11 (92%) patients received bridging therapy after apheresis prior to MCARH109 infusion; all patients were refractory to bridging therapy. There were no dose limiting toxicities. Cytokine release syndrome (CRS) grade 1-3 occurred in 11 (92%) patients with only one patient with grade 3 event; 4 (25%) patients received tocilizumab and 1 (8%) received dexamethasone for the treatment of CRS (Table). There were no neurologic toxicities reported to date; 3 (25%) patients had grade 1 nail changes possibly related to MCARH109 (Table). As of July 28, 2021, all treated patients have been followed for at least 2 weeks (median: 13.0 weeks; range: 2.0-39.1 weeks) and 10 (83%) had at least a minimal response or better (2 responses unconfirmed): 2 minimal response, 3 partial response, 3 very good partial response, 2 stringent complete response (sCR). 5 (56%) of the first 9 patients were minimal residual disease (MRD) negative in the bone marrow by multicolor flow cytometry (sensitivity: 10 -5). 6 (100%) patients with prior BCMA CAR T therapy had a response with 2 patients achieving sCR. We also noted robust MCARH109 expansion in the peripheral blood using qPCR across the first 3 dose levels with available data (peak expansion vector copy number/mL, median: 404,467; range: 44,670- 3,560,000; Table). With a median follow-up of 13 weeks, 9 (75%) patients are progression free and followed without additional therapy. Conclusions: MCARH109 is the first-in-class GPRC5D targeted CAR T cell therapy for MM and has a very manageable safety profile with no serious or unexpected toxicities; this dose escalation study is ongoing with additional patients planned for treatment at higher doses. Efficacy is promising in heavily pre-treated RRMM, reflected in high rates of clinical response as well as MRD-negativity, including at doses as low as 25x10 6 CAR T cells. Clinically important, all 6 patients who relapsed after BCMA CAR T therapy responded to GPRC5D targeted CAR T therapy, including 2 patients who achieved sCR. Figure 1 Figure 1. Disclosures Mailankody: Allogene Therapeutics: Research Funding; Physician Education Resource: Honoraria; Bristol Myers Squibb/Juno: Research Funding; Takeda Oncology: Research Funding; Fate Therapeutics: Research Funding; Jansen Oncology: Research Funding; Evicore: Consultancy; Legend Biotech: Consultancy; Plexus Communications: Honoraria. Shah: Janssen: Research Funding; Celgene/BMS: Research Funding. Lesokhin: pfizer: Consultancy, Research Funding; Iteos: Consultancy; Trillium Therapeutics: Consultancy; Genetech: Research Funding; Serametrix, Inc: Patents & Royalties; bristol myers squibb: Research Funding; Janssen: Honoraria, Research Funding; Behringer Ingelheim: Honoraria. Korde: Medimmune: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Hassoun: Celgene, Takeda, Janssen: Research Funding. Hultcrantz: GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Daiichi Sankyo: Research Funding; Intellisphere LLC: Consultancy; Curio Science LLC: Consultancy. Shah: Amgen: Research Funding; Janssen: Research Funding. Landau: Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Genzyme: Honoraria. Scordo: Angiocrine Bioscience: Consultancy, Research Funding; Omeros Corporation: Consultancy; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; i3 Health: Other: Speaker; McKinsey & Company: Consultancy. Roshal: Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Celgene: Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Landgren: Janssen: Other: IDMC; Janssen: Honoraria; Janssen: Research Funding; Amgen: Honoraria; Celgene: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Dogan: Physicians' Education Resource: Honoraria; Seattle Genetics: Consultancy; Peer View: Honoraria; Takeda: Consultancy, Research Funding; Roche: Consultancy, Research Funding; EUSA Pharma: Consultancy. Giralt: Actinnum: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Park: Autolus: Consultancy; Kite Pharma: Consultancy; PrecisionBio: Consultancy; Minerva: Consultancy; Curocel: Consultancy; Intellia: Consultancy; Amgen: Consultancy; Affyimmune: Consultancy; Innate Pharma: Consultancy; Novartis: Consultancy; Servier: Consultancy; Kura Oncology: Consultancy; Artiva: Consultancy; BMS: Consultancy. Rivière: FloDesign Sonics: Other: Provision of Services; Juno Therapeutics: Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties. Brentjens: BMS: Consultancy, Patents & Royalties, Research Funding; Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; sanofi: Patents & Royalties; Caribou: Patents & Royalties. Smith: Eureka Therapeutics: Consultancy; Fate Therapeutics: Research Funding; Chimeric Therapeutics: Consultancy; Novarits: Consultancy; Sanofi: Patents & Royalties: GPRC5D antibody based therapies; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: CAR T cells for MM. OffLabel Disclosure: MCARH109 is an experimental GPRC5D targeted CART therapy

Background: Autologous CAR T cell therapy targeting the B-cell specific surface antigen CD19 has demonstrated favorable clinical responses in relapsed or refractory (R/B) B-cell lymphomas (BCL). However, despite 40-60% initial complete response (CR) rates, only a subset of patients experience durable remissions, and there is a need to further improve the efficacy of CAR therapies by preventing relapse and attaining a deeper CR. We hypothesized that the redundancy of CD28 and CD3V signaling in a CAR design incorporating all 3 CD3Vimmunoreceptor tyrosine-based activation motifs (ITAMs) might foster counterproductive T cell differentiation and exhaustion, and therefore created a new CD19 CAR construct with calibrated CAR activation potential by mutating 2 of the 3 ITAMs, termed 1XX. In systemic ALL mouse models, 19-28z1XX CAR induced effective tumor eradication at low CAR T cell doses with improved survival compared to conventional 19-28z CAR. Further preclinical studies demonstrated that the enhanced therapeutic benefit resulted from the reduced strength of activation mediated by the 19-28z1XX CAR, achieving a favorable balance of effector and memory functions, thereby enhancing persistence of functional CAR T cells and promoting effective elimination of CD19+ leukemia at lower T cell doses than needed with 19-28z CAR T cells (Feucht J et al. Nat Med 2019). To further improve the persistence of functional CAR T cells, we screened different humanized CD19-directed scFv in the context of a 19-28z1XX CAR design and proved high specificity and functionality of 19-28z1XX CARs containing a novel humanized scFv T2 - termed 19(T2)28z1XX. Study Design and Methods: This study is a single center Phase I clinical trial of 19(T2)28z1XX in patients with R/R B-cell malignancies at Memorial Sloan Kettering Cancer Center (NCT04464200). Key disease eligibility criteria include R/R diffuse large B cell lymphoma (DLBCL), high grade BCL, primary mediastinal BCL, indolent BCL and chronic lymphocytic leukemia (CLL). Patients with prior CD19 CAR therapies are eligible as long as expression of CD19 is confirmed. Key exclusion criteria include ongoing immunosuppression such as systemic GvHD therapy and active CNS disease. The study uses a 3+3 dose-escalation design to identify the maximum tolerated dose for BCL. There are 5 planned flat-dose levels. Patients will receive conditioning chemotherapy consisting of 3 days of fludarabine and cyclophosphamide followed by a single infusion of 19(T2)28z1XX CAR T cells. In the dose-escalation phase, patients with DLBCL, high grade BCL, and primary mediastinal BCL are eligible to participate. Once the recommended phase 2 dose (RP2D) is determined, the study will open to dose expansion phase with two cohorts. Cohort 1 includes DLBCL, high grade BCL and primary mediastinal BCL (i.e. same eligibility criteria as the dose-escalation phase). Cohort 2 will include patients with indolent BCL, CLL, and Richter's transformation. The dose-expansion part of the trial is designed to further characterize the safety, efficacy, and pharmacokinetics of 19(T2)28z1XX CAR in multiple indications. The primary objective of the trial is to evaluate safety and tolerability and determine the recommended Phase 2 dose of 19(T2)28z1XX. Key secondary objectives include evaluation of 19(T2)28z1XX's efficacy and cellular kinetics. Exploratory objectives include assessment of B cell aplasia, and analysis of serum cytokines. The trial has begun enrollment in August 2020. The investigators are hopeful this study will lead to development of improved CD19 CAR T cell therapy with enhanced efficacy and favorable toxicity profiles with lower infused T cell dose. Disclosures Park: AstraZeneca: Consultancy; Servier: Consultancy, Research Funding; Autolus: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy; Minverva: Consultancy; Artiva: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Kite: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Genentech/Roche: Research Funding; Juno Therapeutics: Research Funding; GSK: Consultancy; Intellia: Consultancy; Allogene: Consultancy. Riviere:Fate Therapeutics Inc.: Consultancy, Other: Ownership interest , Research Funding; FloDesign Sonics: Consultancy, Other: Ownership interest; Juno Therapeutics: Other: Ownership interest, Research Funding; Takeda: Research Funding; Atara: Research Funding. Palomba:Genentech: Research Funding; Juno Therapeutics, a Bristol-Meyers Squibb Company: Honoraria, Research Funding; Regeneron: Research Funding; Novartis: Honoraria; Merck: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria. Brentjens:BMS: Research Funding; Gracell Therapeutics: Consultancy; Juno Therapeutics (a Bristol Myers Squibb company): Patents & Royalties. Sadelain:Atara: Patents & Royalties, Research Funding; Fate Therapeutics: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties; Mnemo: Patents & Royalties; Takeda: Patents & Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as conditioning therapy prior to 19(T2)28z1XX CAR T cell administration.

High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) is the standard of care for relapsed or chemorefractory diffuse large B-cell lymphoma (rel/ref DLBCL). Only 50% of patients are cured with this approach. We investigated whether CD19-specific chimeric antigen receptor (CAR) T cells administered following HDT-ASCT may enhance progression-free survival (PFS). Methods: Eligibility for this study includes poor-risk rel/ref aggressive B-NHL chemosensitive to salvage therapy with: 1) FDG-PET (+) or 2) bone marrow involvement. Patients underwent BEAM conditioned HDT-ASCT and followed by 19-28z CAR-T cells on days +2 and +3. Results: Of 15 subjects treated on study, dose-limiting toxicity was observed at both dose levels (5 x106 and 1 x107 19-28z CAR-T/kg). Ten of 15 subjects experienced CAR T cell-induced neurotoxicity and/or cytokine-release syndrome (CRS), which were associated with greater CAR T cell persistence (p=0.05) but not peak CAR T cell expansion. Serum IFN-g elevation (pl0.001) and possibly IL-10 (p=0.07) were associated with toxicity. The 2-year PFS is 30% (95% CI: 20-70%). Two subjects with progression of disease (POD) were CD19 (-) on re-biopsy. Subjects given decreased naive-like (CD45RA+CCR7+) CD4+ and CD8+ CAR T cells experienced superior PFS (p=0.02 and 0.04, respectively). There was no association between CAR T cell peak expansion, persistence or cytokine changes and PFS. Conclusions: 19-28z CAR T cells following HDT-ASCT was associated with a high-incidence of reversible neurotoxicity and CRS. Following HDT-ASCT, effector CD4+ and CD8+ immunophenotypes may improve disease control. Phenotype selection and/or multiple infusions may be the focus of the next clinical trial. This study is registered at www.clinicaltrials.gov as #NCT01840566.

Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 has produced impressive results in treating patients with B-cell malignancies. Although these CAR-modified T cells target the same antigen, the designs of CARs vary as well as several key aspects of the clinical trials in which these CARs have been studied. It is unclear whether these differences have any impact on clinical outcome and treatment-related toxicities. Herein, we review clinical results reflecting the investigational use of CD19-targeted CAR T-cell therapeutics in patients with B-cell hematologic malignancies, in light of differences in CAR design and production, and outline the limitations inherent in comparing outcomes between studies.

While cord blood transplantation (CBT) is an effective therapy for hematologic malignancies, acute graft-versus-host disease (aGVHD) is a leading cause of transplant-related mortality (TRM). We investigated if biomarkers could predict aGVHD and TRM after day 28 in CBT recipients. Day 28 samples from 113 CBT patients were analyzed. Suppressor of tumorigenicity 2 (ST2) was the only biomarker associated with grades II-IV and III-IV aGVHD and TRM. Day 180 grade III-IV aGVHD in patients with high ST2 levels was 30% (95% confidence interval [CI], 18-43) vs 13% (95% CI, 5-23) in patients with low levels (P = .024). The adverse effect of elevated ST2 was independent of HLA match. Moreover, high day 28 ST2 levels were associated with increased TRM with day 180 estimates of 23% (95% CI, 13-35) vs 5% (95% CI, 1-13) if levels were low (P = .001). GVHD was the most common cause of death in high ST2 patients. High concentrations of tumor necrosis factor receptor-1, interleukin-8, and regenerating islet-derived protein 3-α were also associated with TRM. Our results are consistent with those of adult donor allografts and warrant further prospective evaluation to facilitate future therapeutic intervention to ameliorate severe aGVHD and further improve survival after CBT.

Introduction Chimeric Antigen Receptor (CAR) T cells are associated with unique toxicities, including cytokine release syndrome (CRS) and immune effector cells-associated neurotoxicity syndrome (ICANS). Patients (pts) with severe CRS and ICANS exhibit hemodynamic instability and coagulopathy with evidence of endothelial activation and increased blood brain barrier permeability. Increases in inflammatory cytokines and biomarkers of endothelial activation in serum and CSF have been associated with severe CRS and ICANS. The EASIX (Endothelial Activation and Stress Index) score [lactate dehydrogenase (LDH) (U/L) × creatinine (mg/dl) / platelets (PLT) (109 cells/L)] correlates with severe fluid overload and survival in allogeneic transplant pts. Elevated LDH and low PLT levels have been associated with severe ICANS development, and high IL-6 levels are seen in severe CRS and ICANS. We hypothesized that the EASIX and a newly proposed version of it, the modified-EASIX (mEASIX), in which creatinine is replaced by CRP (mg/dL) as an easily available surrogate for IL6, would be associated with CRS and ICANS in CAR T cells pts. Methods We analyzed 2 different populations of adult CAR T cells pts treated at our institution: 1) B-cell acute lymphoblastic leukemia (B-ALL) pts treated with CD1928z CAR T cells from 2010 to 2016 (NCT01044069), and 2) aggressive diffuse large B-cell lymphoma (DLBCL) pts treated with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) after FDA approval starting from 2018. EASIX and mEASIX scores were calculated for each patient daily from start of lymphodepletion conditioning to day +14. A log transformation using base 2 (log2) was applied to all EASIX/mEASIX variables to reduce skew. CRS and ICANS were graded according to the ASTCT grading system. Results 87 pts, B-ALL (n=53) and DLBCL (n=34), were analyzed. According to ASTCT grading, 83% (72/87) experienced CRS and 54% (47/87) developed ICANS, grade ≥3 in 23% (20/87) and 40% (35/87) of pts, respectively. When analyzed by disease, CRS and ICANS rates were 87% (46/53) and 55% (29/53) for B-ALL and 76% (26/34) and 53% (18/34) for DLBCL, respectively. CRS and ICANS were grade ≥3 in 28% (15/53) and 45% (24/53) of B-ALL pts and in 15% (5/34) and 32% (11/34) of DLBCL pts, respectively. Median time of onset of CRS after CAR T cell infusion was day +2 and median onset of ICANS was day +6 for the overall population and the subgroups. High EASIX and mEASIX scores at start of conditioning were both associated with development of any grade CRS [OR=1.81 (95% CI 1.09-3.36) p=0.038 and OR=1.94 (95% CI 1.32-3.38) p=0.005] and grade ≥3 CRS [OR=1.47 (95% CI 1.05-2.29) p=0.049 and OR=1.34 (95% CI 1.07-1.80) p=0.024], respectively (Table). Following CAR T cell infusion, high scores of both EASIX [OR=1.60 (95% CI 1.12-2.43) p=0.017] and mEASIX [OR=1.32 (95% CI 1.07-1.69) p=0.014] on day +1 were associated with development of grade ≥3 CRS. Moreover, both high EASIX [OR=1.43 (95% CI 1.08-1.96) p=0.018] and mEASIX [OR=1.29 (95% CI 1.07-1.60) p=0.010] scores on day +3 were associated with grade ≥3 ICANS. When analyzed by disease, results were confirmed for severe CRS and ICANS in B-ALL patients, while in the DLBCL group only mEASIX at start of conditioning and at day +1 was associated with development of any grade CRS. EASIX and mEASIX scores were not associated with response rates to CAR T cells therapy. Conclusions EASIX and mEASIX scores calculated at baseline (before lymphodepletion) are associated with development of CRS and severe CRS. Moreover, both high EASIX and mEASIX scores on day +1 and day +3 are associated with occurrence of grade ≥3 CRS and grade ≥3 ICANS, respectively. We conclude that EASIX and mEASIX, as markers of endothelial damage and inflammation, could be useful as early predictors in guiding treatment decisions before the onset of severe symptoms. Table Disclosures Batlevi: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy. Giralt:Miltenyi: Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy, Research Funding; Johnson & Johnson: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Actinium: Consultancy, Research Funding; Kite: Consultancy. Palomba:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Noble Insights: Consultancy; Seres Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; STRAXIMM: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Evelo: Equity Ownership; MSK (IP for Juno and Seres): Patents & Royalties; Hemedicus: Speakers Bureau; Merck & Co Inc.: Consultancy. Santomasso:Kite/Gilead: Consultancy; Juno/Celgene: Consultancy; Novartis: Consultancy. Sauter:GSK: Consultancy; Kite/Gilead: Consultancy; Celgene: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Genmab: Consultancy; Precision Biosciences: Consultancy. Scordo:Angiocrine Bioscience, Inc.: Consultancy; McKinsey & Company: Consultancy. Shah:Amgen: Research Funding; Janssen Pharmaceutica: Research Funding. Park:Allogene: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Autolus: Consultancy; GSK: Consultancy; Incyte: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Takeda: Consultancy. Perales:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Miltenyi: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees.

Background: CAR T therapy is FDA approved for specific relapsed/ refractory (R/R) B cell lymphomas and acute lymphoblastic leukemia (ALL), and clinical trials are ongoing for R/R multiple myeloma (MM). Cytopenias have been observed post-CAR T, yet there is minimal data delineating the pathobiology and trends. We report the largest series to our knowledge thus far, of hematological recovery and factors affecting count recovery after CAR T. Methods: We retrospectively reviewed adult patients who received CAR T for R/R B cell lymphomas after FDA approval and those treated for R/R B cell ALL (NCT01044069) and MM (NCT03070327) at our center. Blood counts were collected for up to 12 months or until censored for relapse, progression or initiation of chemotherapy/ conditioning for autologous or allogeneic stem cell transplantation (HCT)/ subsequent treatment with CAR T. Only patients with follow-up > 30 days were included. "Recovery" for the respective blood count was defined as hemoglobin > 8g/dL and platelets > 50,000/µL without transfusion support in 2 weeks and 1 week, respectively; absolute neutrophil count (ANC) > 1,000/µL and white cell count (WBC) > 3,000/µL without growth factor support in 2 weeks. "Normalization" was defined as normal range for the laboratory; hemoglobin > 11.2g/dL in women and 12.5g/dL in men, platelets > 160,000/µL, ANC > 1,500/µL and WBC > 3,000/µL without transfusion support as above. "Complete count recovery" refers to recovery per above criteria in all 4 counts. Categorical variables were compared using Fisher's exact test and continuous variables using the Wilcoxon rank-sum test. Results: Eighty three patients were included (Table 1). Using the noted nadir values, grade 1-2 and 3-4 anemia was seen in 22% and 78%, thrombocytopenia in 29% and 66%, neutropenia in 3% and 96% while leucopenia in 0% and 100%, respectively. During the follow-up, 66% patients received packed red cell transfusion, 52% received platelet transfusion and 62% received growth factor support. By 1 month (n=83), recovery of hemoglobin, platelets, ANC and WBC was noted in 61%, 51%, 33% and 28%, respectively. At 3 months (n=41), these respective percentages were 93%, 90%, 81% and 59%. All patients had recovered hemoglobin and platelet count by 4 months (n=17), and ANC by 9 months (n=14). By 3 months, normalization of hemoglobin, platelets, ANC and WBC was noted in 39%, 34%, 71%, and 39%, respectively. Upon examination of potential variables in a univariate model, lack of recovery of hemoglobin, platelets, ANC and complete counts recovery at 1 month was statistically significantly associated with type of CAR construct, higher grade (grade 3-4 vs grade 1-2 vs none) cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) as well as a higher peak CRP and ferritin (data for complete count recovery in Table 2). Additionally, lack of hemoglobin recovery at 1 month was associated with lymphodepletion using high dose cyclophosphamide (recovered vs no recovery in 58% vs 42%, p=0.01) and diagnosis of ALL (65% vs 35%, p = 3 prior lines of therapy (78% vs 22%, p = 0.04). At 3 months, absence of complete count recovery was associated only with the CAR T construct utilized. A multivariate logistic regression model resulted in wide confidence intervals due to small size of subgroups, hence leading to unreliable point estimates (data not shown). Conclusions: Our study shows that blood counts recover in most patients who have not progressed or received additional therapy by 3 months post-CAR T. The association of count recovery with severity of CRS, ICANS as well as inflammatory marker levels indicates that inflammatory response post-CAR T influences hematological recovery in these patients. The association of count recovery and CAR construct can be influenced by underlying diagnosis as specific CAR constructs were used for specific diagnosis. Since patients with no disease response were excluded and were censored at progression, these effects are less likely to be affected by disease response; however the association of depth of response could not be evaluated in this study. These results warrant future studies to understand underlying mechanisms of inadequate recovery. Disclosures Scordo: McKinsey & Company: Consultancy; Angiocrine Bioscience, Inc.: Consultancy. Sauter:Kite/Gilead: Consultancy; Precision Biosciences: Consultancy; Genmab: Consultancy; Celgene: Consultancy; GSK: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy. Santomasso:Novartis: Consultancy; Kite/Gilead: Consultancy; Juno/Celgene: Consultancy. Palomba:Noble Insights: Consultancy; Hemedicus: Other: Immediate Family Member, Speakers Bureau ; Evelo: Other: Immediate family member, Equity Ownership; MSK (IP for Juno and Seres): Other: Immediate Family Member, Patents & Royalties - describe: intellectual property rights ; Kite Pharmaceuticals: Other: Immediate Family Member, Membership on an entity's Board of Directors or advisory committees; Merck & Co Inc.: Other: Immediate Family Member, Consultancy (includes expert testimony); Seres Therapeutics: Other: Immediate Family Member, Equity Ownership and Membership on an entity's Board of Directors or advisory committees; STRAXIMM: Other: Immediate Family Member, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Shah:Amgen: Research Funding; Janssen: Research Funding. Batlevi:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Brentjens:Celgene: Consultancy; JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding. Park:Takeda: Consultancy; Novartis: Consultancy; Kite Pharma: Consultancy; Incyte: Consultancy; Allogene: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Autolus: Consultancy; GSK: Consultancy. Perales:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Miltenyi: Research Funding; MolMed: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mailankody:Celgene: Research Funding; Juno: Research Funding; Janssen: Research Funding; Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria.

Introduction: Autologous chimeric antigen receptor (CAR) T cell therapy has shown to be effective in CD19+ relapsed or refractory (R/R) B-ALL patients (pts). However, relapses are common and the role of post-CAR allogeneic hematopoietic stem cell transplant (alloHSCT) remains unclear, particularly in adults with relapsed B-ALL. We previously reported that there was no survival benefit with post-CAR alloHSCT in overall adult ALL population (Park J et al. NEJM 2018), but it is unclear whether there is a subset of pts who may benefit the most from post-CAR alloHSCT. Therefore, we sought to identify clinical features that are associated with a better long-term survival in adult B-ALL pts treated with CD19 CAR T cells followed by alloHSCT. Methods: We performed a retrospective chart review of adult pts with R/R ALL treated with 19-28z CAR T therapy at MSKCC (NCT01044069), with a particular focus on pts who achieved minimal residual disease (MRD) negative CR to the CAR therapy and subsequently proceeded to alloHSCT. Clinical data was updated with the data cutoff date of July 15, 2019. We examined the association between overall survival and the following clinical factors: impact of age, prior HSCT status, presence of Philadelphia chromosome (Ph), disease status at the time of CAR therapy (MRD vs. morphologic), severity of cytokine release syndrome (CRS) and neurotoxicity (NTX) experienced during CAR therapy. Survival was calculated from the time of CAR T cell infusion. Continuous variables were analyzed univariately using cox PH models. Categorical variables were analyzed using Kaplan Meier methodology and log rank tests. Results: Of the 53 pts who received 1928z CAR T cells, 16 pts proceeded to alloHSCT after achieving MRD negative CR to the CAR therapy. The median age of these pts was 38 (range, 23-66), and baseline disease and pt characteristics as well as response and toxicity to CAR T therapy are listed in Table 1. Of the 16 pts, 6 pts remain alive in remission; 4 pts died of relapse (median time to relapse: 104.5 days, 57-194), and 6 pts died of transplant-related mortality (TRM) in remission (n=4, infection; n=1, pulmonary VOD; n=1, respiratory failure of unclear etiology). Detailed disease and treatment characteristics by the outcome categories are listed in Table 2. For those pts who are alive in remission, the median survival is 47.5 months (range, 39.7-92.4). Among the examined clinical variables, we found younger age and no severe NTX (i.e. grade 0-2) after CAR therapy to be significantly associated with improved overall survival (p=0.014 and 0.05, respectively) but prior lines of therapy, time to HSCT, prior HSCT, Ph chromosome, disease status at the time of T cell therapy and severity of CRS did not impact the survival after alloHSCT. Conclusions: In this retrospective analysis, we found that younger age and no severe NTX to CD19 CAR therapy were associated with improved overall survival following alloHSCT. Number of relapses following alloHSCT (4 of 16 pts) appears to be lower compared to the previously reported number of relapse in the adult ALL cohort without alloHSCT (17 of 26 pts). However, the occurrence of TRM due to infection is high, likely a reflection of heavily pretreated pt population and possibly further immunosuppression from CAR + alloHSCT. The link between severe NTX and worse survival is unclear but could be related to prolonged corticosteroid use and high disease burden. While no definitive conclusion can be drawn due to a small sample size, our data suggests a certain subset of adult pts with R/R B-ALL may benefit more from alloHSCT as a consolidation therapy following CD19 CAR T cells, and acute and late infectious complications following alloHSCT should be carefully monitored. These findings should be validated prospectively and compared between different CAR constructs. Disclosures Park: Allogene: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Autolus: Consultancy; GSK: Consultancy; Incyte: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Takeda: Consultancy. Riviere:Fate Therapeutics: Consultancy; Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents & Royalties; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy.

Adoptive cell therapy with tumor-targeted T cells is a promising approach to cancer therapy. Enhanced clinical outcome using this approach requires conditioning regimens with total body irradiation, lymphodepleting chemotherapy, and/or additional cytokine support. However, the need for prior conditioning precludes optimal application of this approach to a significant number of cancer patients intolerant to these regimens. Herein, we present preclinical studies demonstrating that treatment with CD19-specific, chimeric antigen receptor (CAR)–modified T cells that are further modified to constitutively secrete IL-12 are able to safely eradicate established disease in the absence of prior conditioning. We demonstrate in a novel syngeneic tumor model that tumor elimination requires both CD4+ and CD8+ T-cell subsets, autocrine IL-12 stimulation, and subsequent IFNγ secretion by the CAR+ T cells. Importantly, IL-12–secreting, tumor-targeted T cells acquire intrinsic resistance to T regulatory cell–mediated inhibition. Based on these preclinical data, we anticipate that adoptive therapy using CAR-targeted T cells modified to secrete IL-12 will obviate or reduce the need for potentially hazardous conditioning regimens to achieve optimal antitumor responses in cancer patients.

Introduction: BCMA targeted CAR T cell therapy has shown promising results in patients with relapsed/refractory multiple myeloma (MM). Herein, we report on the safety and efficacy of MCARH171, a second generation, human derived BCMA targeted autologous 4-1BB containing CAR T cell therapy, including a truncated epidermal growth factor receptor safety system (Smith EL. Mol Ther 2018). Methods: This is a phase I first in human, dose escalation trial of MCARH171. Patients received conditioning chemotherapy with cyclophosphamide (Cy) 3 gm/m2 as a single dose or fludarabine 30 mg/m2 daily and Cy 300 mg/m2 daily for 3 days followed by MCARH171 infusion in 1-2 divided doses. The trial followed a standard 3+3 design with 4 dose levels where patients received the following mean doses per cohort: (1) 72x106, (2) 137x106, (3) 475x106, (4) 818x106 viable CAR+ T cells. The primary objective was to demonstrate safety, and secondary objectives included efficacy and expansion, and persistence of CAR T cells using PCR from the peripheral blood. The last accrued patient received MCARH171 on Dec 6, 2017 and the data cut-off is July 16, 2018. The study is closed to accrual. Results: 11 patients with relapsed and/or refractory MM were treated. Median number of prior lines of therapy was 6 (range: 4-14), and all patients received prior therapy with a proteasome inhibitor, IMiD, anti-CD38 monoclonal antibody, and high dose melphalan/stem cell transplant. Nine (82%) patients had high-risk cytogenetics and 9 (82%) were refractory to their immediate prior line of treatment. One patient was not evaluable for DLTs given the need for early radiation and steroids for impending spinal cord compression by tumor. There are no DLTs reported. Cytokine release syndrome (CRS) grade 1-2 occurred in 4 patients (40%), grade 3 occurred in 2 (20%), and there was no grade 4-5 CRS. Grade 2 encephalopathy occurred in 1 patient (10%) in the setting of high fevers which resolved in less than 24 hours. There was no grade 3 or higher neurotoxicity observed. Tocilizumab was administered to 3 patients; 2 in cohort 2, and 1 in cohort 3. Laboratory values correlating with CRS reaching grade 3 or requiring Tocilizumab (N=4) compared to those with no or milder CRS (N=6) included peak CRP (mean: 28.5 vs 4.6 mg/dL, p Overall response rate was 64% and the median duration of response was 106 days (range: 17 to 235 days). The peak expansion and persistence of MCARH171 as well as durable clinical responses were dose dependent. Patients who were treated on the first two dose cohorts (≤150 X106 CAR T cells) had a lower peak expansion in the peripheral blood (mean 14,098 copies/µL; N=6), compared to patients who were treated on the third or fourth dose cohort 3-4 (≥450 X106 CAR T cells; N=5), where the mean peak expansion was 90,208 copies/µL (p6 months compared to only 1 of 6 (16.7%) patients who received lower doses. Two patien have ongoing responses (VGPR) at 7.5+ and 10+ months of follow up. To normalize for dose administered we compared the pharmacokinetics of only patients treated at dose levels 3-4 ( ≥450 X106 CAR T cells). Here, we demonstrate that peak expansion correlated to clinical efficacy, with the 3 durable responders all having peak expansion >85,000 copies/µL (mean: 131,732 copies/µL); compared to transient responders, where the maximum peak expansion was 33,213 copies/µL (mean: 27,922; Figure 1). Conclusions: MCARH171 has an acceptable safety profile with no DLTs reported. A dose-response relationship with toxicity was not clearly observed, as noted by distribution of tocilizumab use across dose cohorts. However, a dose-response relationship was observed with promising clinical efficacy at dose levels of ≥450 X106 CAR T cells. Controlling for dose level, peak expansion correlated with durability of response. These results further support the development of CAR T cells for heavily pre-treated patients with relapsed and refractory MM. Disclosures Mailankody: Janssen: Research Funding; Takeda: Research Funding; Juno: Research Funding; Physician Education Resource: Honoraria. Korde:Amgen: Research Funding. Lesokhin:Takeda: Consultancy, Honoraria; Squibb: Consultancy, Honoraria; Janssen: Research Funding; Genentech: Research Funding; Serametrix, inc.: Patents & Royalties: Royalties; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding. Hassoun:Oncopeptides AB: Research Funding. Park:Juno Therapeutics: Consultancy, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; AstraZeneca: Consultancy; Adaptive Biotechnologies: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Shire: Consultancy. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Palomba:Pharmacyclics: Consultancy; Celgene: Consultancy. Riviere:Fate Therapeutics Inc.: Research Funding; Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Landgren:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding.

Introduction: One of the main complications of adoptive T cell therapy (ACT) is the en-masse activation of tumor-reactive T cells inducing a large release of cytokines followed by activation of other immune cells leading to adverse events. These are classified as a cytokine release syndrome (CRS) or neurotoxicity described as a CAR T Related Encephalopathy Syndrome (CRES). Several biomarkers have been associated with CRS and/or neurotoxicity such as LDH, ferritin and CRP. Cytokines have also been associated with CRS and and/or CRES, but present approaches rely on retrospective study of collected biomarkers. Here, we report the results of cytokine analysis using a point of care (POC) device to predict immune-related toxicities in patients with relapsed/refractory (R/R)DLBCL treated with axicabtagene ciloleucel (axi-cel). Methods: Patients with R/R DLBCL treated with commercial axi-cel were included in this study. Baseline serum samples were collected prior to lymphodepleting chemotherapy and then daily during hospitalization. To select which cytokines to monitor, we retrospectively analyzed 38 serum cytokines in a cohort of 53 patients with R/R B cell acute lymphoblastic leukemia (B-ALL) who were treated with 19-28z CAR T cells. The patients were divided into those requiring treatment with tocilizumab and/or steroids versus those who did not require treatment. We observed several cytokines, including IL-2, IL-6, IL-15 and IFNg, which were significantly elevated in patients with CRS and/or CRES requiring treatment (Figure 1a). Based on this analysis and results of published studies, eight serum proteins were selected in our study including IL-1b, IL-2, IL-6, IL-15, IFNg, TNFa, and angiopoietin-1 &2. We monitored these proteins using a POC device that allows for rapid daily monitoring with a turnaround time of two hours. We established that the results from the POC device strongly correlate with a current gold standard device(Luminex), which has a typical two day turn around time. CRS and CRES were prospectively graded using revised Lee criteria (Lee et al Blood 2014) and the CARTOX group (Neelapu et al. NRCO 2017) respectively by an experienced clinical team and confirmed by chart review retrospectively. Results: A total of 20 patients with R/R DLBCL treated with commercial axi-cel were identified. Median age 64 years ( range 43-73) with 80% male.In our cohort, grades 1-3 CRS were observed in 45%, 40% and 5% respectively. There were no observed grade 0 or grade 4 CRS. There were two patients (10%) who died in the setting of severe toxicity. Patients with grade 5 CRS had higher levels of IL-6 and angiopoietin 2/angiopoietin 1 ratio at day one, which correlated with severity of toxicity r=0.52 (p= 0.039) , and r=0.53 (p=0.033) respectively (Fig. 1b). Furthermore, patients with high grades CRS had elevated levels of IL-15 at day seven (r=0.83, p=0.006). The majority of patients (55%) had grade 1-2 CRES.There were no significant correlations between serum cytokine levels and CRES or between those who required tocilizumab/steroids vs. those who did not, likely due to the small sample size. In select cases, daily monitoring of cytokines using the POC device provided clinical insight that wasn't evident from standard biomarkers. For example, one patient who developed delayed CRS had high serum levels of IL-6 but did not have elevated levels of CRP(Fig.1c). Discussion: In this analysis of 20 patients, we observed a correlation between severe CRS and elevated serum cytokine levels of IL-6 and angiopoietin 2/angiopoietin 1 ratio at day one suggesting that these biomarkers may be utilized to predict severe toxicity in patients treated with ACT. While this study is limited by small sample size, our observations correlate with previously published biomarkers data in patients enrolled in clinical trials. To our knowledge this is the first reported cytokine data using commercial axi-cel. Monitoring of cytokines using a POC device is feasible and will be useful clinically. High risk patients may be identified early and help guide intervention in real time, for example day one elevated IL-6 levels might inform earlier use of tocilizumab. We continue to enroll patients to validate cytokines as predictive biomarkers with the goal of informing the development of preventative strategies to mitigate CAR T cell therapy immune related adverse events. Disclosures Locke: Cellular BioMedicine Group Inc.: Consultancy; Kite Pharma: Other: Scientific Advisor; Novartis Pharmaceuticals: Other: Scientific Advisor. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Park:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Kite Pharma: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Shire: Consultancy. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Introduction Cellular therapy with chimeric antigen receptor (CAR) T cells has fundamentally changed the treatment of many cancers. Unfortunately, not all patients who receive this therapy have a favorable response. Additionally, some patients may develop toxicity due to cytokine release syndrome (CRS) or neurotoxicity. Recent studies have found a relationship between the intestinal microbiome and the response to immunotherapy with checkpoint blockade. We propose the intestinal microbiota as a factor that influences the efficacy and toxicity of CAR T cells. We hypothesize that the differences in outcomes of patients who receive CAR T cells are related to the composition of their intestinal microbiota at baseline. We report a single-center analysis of pre-CAR T cell infusion microbiota composition. Methods We collected stool samples from recipients of CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC). A baseline sample was collected prior to CAR T cell infusion. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the operational taxonomic units (OTUs) were classified using the NCBI Reference Sequence Database. Clinical response to assess efficacy was classified as either complete response (CR) or no complete response. Given the sample size, toxicity was pooled to encompass Grade 1 to 4 CRS and Grade 1 to 4 neurotoxicity. Linear discriminant analysis effect size (LEfSe) was used to identify microbial biomarkers for efficacy and toxicity between groups using relative abundances with a linear discriminant analysis score threshold >2.5. Results We analyzed 24 baseline samples from 24 patients treated at MSKCC. The patients were all adult recipients of cellular therapy with CAR T cells. The patients varied in conditioning regimen, CAR construct and underlying diagnosis, which included solid tumors and hematologic malignancies. First, we assessed the 16S relative abundance of the intestinal microbiota of the patients at baseline. We found that the composition of the microbiota prior to CAR T cell infusion was diverse, as defined by an Inverse Simpson >4 in all of the patients, although the level of diversity amongst the patient samples varied (Fig A). An assessment of the efficacy of CAR T cells with LEfSe analysis found increased abundance in several families of the Clostridiales order (Firmicutes phylum), including Oscillospiraceae, Ruminococcacaeae, and Lachnospiraceae, in those patients who achieved a CR. For the patients who did not achieve a CR, we found an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Patients who experienced toxicity, either CRS or neurotoxicity, had an increased abundance of families within the Clostridiales or Lactobacillales order (Firmicutes phylum), which included Lachnospiraceae and Lactobacillaceae. Finally, patients who did not experience toxicity also had an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Conclusion We demonstrate that our subset of patients had diverse microbial composition prior to receiving CAR T cell therapy despite the fact that many of them were heavily pre-treated. Additionally, we observe the abundance of the family Lachnospiraceae in the patients who achieved a CR and those who experienced toxicity. Many Lachnospiraceae are butyrate producers, whose presence has been found to be protective against Clostridium difficile infection in recipients of allogeneic hematopoietic cell transplant but whose abundance is lower in colon cancer. Conversely, we observe an abundance of the family Peptostreptococcaceae in patients who did not achieve a CR or who did experience toxicity. Peptostreptococcaceae has been found to be more abundant in the intestines of patients with colon cancer. Of note, the intestinal micriobiota that we identify are not congruent with the specific bacteria that have been found to promote anti-tumor immunity to checkpoint blockade. Our data suggests a role for the intestinal microbiota in mediating the response to CAR T cells and proposes that the baseline microbial composition may correlate with efficacy and toxicity. Further studies will investigate biochemical mechanisms to understand the interplay of the intestinal microbiota and the immune system to improve patient outcomes following CAR T cell therapy. Disclosures Park: Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; AstraZeneca: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Shire: Consultancy. O'Cearbhaill:Juno: Research Funding. Mailankody:Juno: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Physician Education Resource: Honoraria. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Palomba:Pharmacyclics: Consultancy; Celgene: Consultancy. Riviere:Fate Therapeutics Inc.: Research Funding; Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Background: Chimeric antigen receptor-modified (CAR) T cells have the potential to provide durable clinical benefit in patients with several relapsed or refractory hematologic malignancies. We aimed to characterize institutional resources utilized (other than T cell collection and CAR T cell manufacturing and infusion) around the time of CAR T cell administration. Methods: Adult patients treated on selected investigator-initiated clinical trials of CAR T cell therapy at Memorial Sloan Kettering Cancer Center were identified from the institutional database. Utilization data was collected from the start of admission for CAR T cell infusion through the end of the initial admission for infusion or through 30 days following initial CAR T cell infusion, whichever was longer. The data were sorted by disease type and into the categories of encounters, lab work, radiology, medications, and other diagnostic testing. Descriptive statistics were used to analyze the data. Results: We identified 106 patients on 4 clinical trials receiving inpatient CAR T cell infusions between 6/2007 to 4/2018, with 56 patients (53%) having B-cell acute lymphoblastic leukemia (ALL), 37 (35%) chronic lymphocytic leukemia (CLL) or B-cell non-Hodgkin lymphoma (NHL), and 13 (12%) multiple myeloma (MM). The median age was 53 years (range 22-77), 45 years (range 22-74), 64 years (range 35-77), and 58 years (range 43-68) for the total population, and the ALL, CLL/NHL, and MM groups, respectively, and 65%, 75%, 41%, and 31% were male, respectively. The median length of stay for the admission during which CAR T cells was given was 23 days (range 4 -133), with ALL patients admitted longer (Figure 1). Intensive care unit (ICU) days were limited with a range of 0-43 days, though 43 (41%) spent at least one day in the ICU. Of note, some protocols required infusion of the CAR T cells to be in the ICU. ICU admissions for ALL patients were than for other histologies longer (median 9 days vs 4 days). Outpatient clinic visits through day 30 post CAR T cell infusion occurred in 57 (53%) patients, with more of these in the CLL/NHL patients (median of 2 visits, range 1-4). As expected, laboratory and radiology studies accounted for a large portion of resource utilization with a total of 62,953 laboratory panels and 1,190 radiology studies done during the study time frame. Fourteen percent of the labs were complete blood counts, basic or comprehensive metabolic panels, or liver function tests. For the total population, ALL, CLL/NHL, MM, there were a median of 63.5 (range 13-368), 91.5 (21-368), 47 (13-167), and 51 (range 38-113) of these panels done per patient during the time frame, respectively. Blood cultures accounted for 1.3% of the total laboratory tests. Among the radiology studies, 25% were CT scans, 10% MRIs, 7% PET scans, 5% ultrasounds, and 53% x-rays, with differences in patterns of use by disease type (Figure 2). Cardiac testing (echocardiographs & electrocardiograms) was done in 104 (98%, total 634 tests). Electroencephalogram was performed in 18 patients (17%, total 18 tests). There were 173 bone marrow aspirations/biopsies in 88 patients (83%) and 71 lumbar punctures in 38 patients (36%), many of which were potentially done for disease assessment rather than toxicity management. Finally, 41,331 units of medications were given in this time frame, of which chemotherapy was 328 units (0.8%). The median medication units per patient was 255 (range 35-2091), 423 (range 35-2091), 200 (range 41-1190), and 207 (range 90-666) for the total population, and the ALL, CLL/NHL, and MM patients groups, respectively. Thirty-two doses of tocilizumab were given to 25 patients (24%), with ALL patients receiving 23 of those doses (72%). Conclusion: While providing potential clinical benefit, CAR T cell therapy utilizes resources across the therapeutic spectrum, and increasing use of this therapeutic modality can create challenges in institutional resource capacity. Identifying these resources will allow for better care delivery and allocation of funds. Further refinement of CAR T cell products and improvements in CAR T cell-related toxicity management may permit safer delivery of this therapy and reduce costs per patient. Additional analysis of resource utilization among patients treated with commercial CAR T cell products, as well as comparison with alternative therapies and cost-effectiveness analysis, is warranted. Disclosures Shah: Amgen: Research Funding; Janssen: Research Funding. Park:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Shire: Consultancy; AstraZeneca: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Kite Pharma: Consultancy; Juno Therapeutics: Consultancy, Research Funding. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Palomba:Pharmacyclics: Consultancy; Celgene: Consultancy. Younes:Genentech: Research Funding; BMS: Honoraria, Research Funding; Pharmacyclics: Research Funding; Abbvie: Honoraria; Merck: Honoraria; Takeda: Honoraria; J&J: Research Funding; Incyte: Honoraria; Janssen: Honoraria, Research Funding; Celgene: Honoraria; Roche: Honoraria, Research Funding; Curis: Research Funding; Astra Zeneca: Research Funding; Novartis: Research Funding; Bayer: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria. Geyer:Dava Oncology: Honoraria. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Mailankody:Physician Education Resource: Honoraria; Janssen: Research Funding; Juno: Research Funding; Takeda: Research Funding. Perales:Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Merck: Other: Personal fees; Takeda: Other: Personal fees; Abbvie: Other: Personal fees; Novartis: Other: Personal fees. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Bach:Vizient: Other: Personal Fees; Hematology Oncology Pharmacy Assoc: Other: Personal Fees; Excellus Health Plan: Other: Personal Fees; Gilead: Other: Personal Fees; Foundation Medicine: Other: Personal Fees; JMP Securities: Other: Personal Fees; Janssen: Other: Personal Fees; Grail: Other: Personal Fees; American Society for Hospital Pharmacy: Other: Personal Fees; Kaiser Permanente: Research Funding; Third Rock Ventures: Other: Personal Fees; WebMD: Other: Personal Fees; Anthem: Other: Personal Fees; Goldman Sachs: Other: Personal Fees; Novartis: Other: Personal Fees; Defined Health: Other: Personal Fees.

Background: CD19-specific 2nd generation chimeric antigen receptor (CAR) T cell therapy with either CD28 or 41BB co-stimulatory domain has demonstrated complete remission (CR) rates of 30-50% in relapsed or refractory (R/R) NHL and 20-30% in CLL. The lesser activity of CD19 CAR T cells in CLL and NHL relative to ALL may be in part due to diminished intrinsic T cell quality or greater CAR T cell inhibition in the tumor microenvironment. We previously demonstrated that 19-28z CAR T cells that express 4-1BBL achieve greater proliferation, IL-2 secretion and persistence compared to 19-28z or 19-4-1BBz CAR T cells (Zhao et al., Cancer Cell, 2015). Herein, we report on adult patients (pts) with CLL or NHL treated with escalating doses of autologous 19-28z/4-1BBL+ CAR T cells (NCT03085173). Methods: Pts with R/R NHL (DLBCL, follicular lymphoma (FL), transformed FL (tFL), Waldenström's macroglobulinemia (WM)) and CLL including Richter's transformation were eligible for the trial. Pts received conditioning chemotherapy of cyclophosphamide (Cy) alone or combined with fludarabine (Flu) followed by escalating doses of CAR T cells. CAR T cells were administered at dose level (DL) 1 (1x105 CAR T cells/kg), DL2 (3x105 CAR T cells/kg), DL 3 (1x106 CAR T cells/kg), and DL4 (3x106 CAR T cells/kg). The primary objective of the study was to evaluate safety of autologous 19-28z/4-1BBLCAR T cells. Secondary objectives included rates of overall response. Results: 25 pts were enrolled with R/R CLL (n=9), de novo DLBCL (n=6), tFL (n=3), FL and WM (n=4), and Richter's transformation (n=3). Median age of the pts was 70 (range, 53-81), and median number of prior treatments was 5 (range, 2-17). All 10 pts with CLL progressed on ibrutinib and 9 pts were refractory to venetoclax. 3 pts received DL1 CAR T cells, 3 pts DL2 CAR T cells, 3 pts DL3 CAR T cells and remaining 16 pts received DL4 CAR T cells. Of the 9 de novo DLBCL and tFL pts, all were >70 years old, 7/9 were refractory with progressive disease as best response to any line of chemotherapy or relapsed ≤12 mo after ASCT or any line of therapy. Six of 9 pts received R-GemOx as bridging chemotherapy with 2/6 progressing on R-Gemox prior to CAR T cell therapy. 3 pts received conditioning chemotherapy with Cy alone, and 22 pts received Flu + Cy conditioning chemotherapy. No dose-limiting toxicity has been observed. Of the 25 treated pts, 24 pts are at least 2 weeks from T cell infusion and evaluable for toxicity. Overall, 16 pts experienced CRS (67%), all of which were grade 1 (n=13) and grade 2 (n=3). No pts experienced severe CRS. 8 pts (33%) experienced any grade neurotoxicity (NTX), including 6 pts with grade 1-2 and 2 pts with grade 3 NTX. No cerebral edema was observed, and all NTX were transient and reversible. 2 pts (8%) received one dose of tocilizumab and 4 pts (16%) received corticosteroid for NTX. 21 pts had at least 4 weeks of follow-up and were evaluable for response. 12 of 21 pts (57%) achieved CR, including 7 of 8 pts (88%) with large cell lymphoma, 2 of 2 pts with FL, 2 of 9 pts (22%) with CLL and 1 of 2 pts with Richter's transformation (Figure). CR was observed in pts with ≥10cm bulky lymph nodes (n=1), extra nodal disease involving bone, muscle, perineural and CNS (n=3), and extensive bone marrow involvement ≥80% (n=3). With a median follow-up of 93 days (range, 30 - 439), 11 of the 12 CR pts remain in CR. Peak CAR T cell expansion occurred at a median of 9 days after CAR T cell infusion (range, 2-82). While analysis of CAR T cell persistence is ongoing, CAR T cell detection beyond 160 days has been noted. Conclusions: Treatment with 19-28z/41BBL armored CAR T cells appears to be safe. No severe CRS was observed and severe NTX occurred in 8% of the pts with no case of cerebral edema. The overall CR rate of 57% is encouraging with 11 of the 12 pts remaining in CR at the time of this report. CR rates were higher in pts with large cell lymphoma (88%) compared to CLL (22%), though most of CLL pts received lower dose of CAR T cells (7 pts at DL1-3 vs. 2 pts at DL4). Pts with CLL may require higher doses of CAR T cells or incorporation of the CAR therapy in earlier lines of treatments. Detailed cytokine and CAR T cell expansion analysis in comparison to our previous cohort of pts treated with the 2nd generation 1928z CAR T cells will be presented. Figure. Figure. Disclosures Park: Adaptive Biotechnologies: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Shire: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Pfizer: Consultancy. Palomba:Celgene: Consultancy; Pharmacyclics: Consultancy. Riviere:Fate Therapeutics Inc.: Research Funding; Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Furman:Pharmacyclics LLC, an AbbVie Company: Consultancy; Acerta: Consultancy, Research Funding; Genentech: Consultancy; Gilead: Consultancy; Incyte: Consultancy, Other: DSMB; Loxo Oncology: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Sadelain:Fate Therapeutics Inc.: Research Funding; Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Early clinical results using BCMA targeted CAR T cell therapies for advanced multiple myeloma (MM) have shown promise. However, BCMA expression can be variable, and BCMA downregulation has been correlated with relapse (Brudno J. JCO. 2018; Cohen A. ASH. 2017). Targeting multiple antigens may enhance response durability. We report that the orphan seven transmembrane G protein coupled receptor, GPRC5D, is an attractive additional target for CAR T cell therapy of MM. GPRC5D mRNA expression was previously identified in bone marrow cells from patients with MM; however its protein expression could not be detected with available FACS reagents (Frigyesi I. Blood. 2014). We evaluated 83 primary marrow samples by quantitative immunofluorescence (Q-IF) for CD138, BCMA, and GPRC5D. In 98% of the samples, CD138+ cells expressed surface GPRC5D. In most samples, the majority of CD138+ cells expressed both BCMA and GPRC5D, however, in several cases the dominant CD138+ population expressed only BCMA or GPRC5D, with GPRC5D expression independent of BCMA across samples (R2=0.156; Figure 1). The potential for "on target/off tumor" binding by targeting GPRC5D was evaluated by screening 30 essential normal tissue types by IHC (n=3 donors/type) followed by validation of any positive findings by RNA in situ hybridization and quantitative PCR. Results in non-plasma cell normal tissue were consistent with prior reports of GPRC5D off target expression restricted to cells from the hair follicle, a potentially immune privileged site. We developed GPRC5D-targeted CARs considering immunogenicity, spacer length, and tonic signaling. To minimize potential anti-CAR immunity, a human B cell-derived phage display library was screened. Seven diverse and highly specific human single chain variable fragments (scFvs) were identified. 42 CARs were derived from the 7 scFvs by modifying scFv orientation (VH/VL; VL/VH) and incorporating a short, medium, or long IgG4 based spacer. To monitor CAR-mediated signaling, we transduced each CAR into a Jurkat reporter line with RFP inserted in-frame, downstream of endogenous NR4A1 (Nur77). Nur77 expression is an immediate-early, specific marker of CD3z signaling (Ashouri J. J Immunol. 2017). Using this reporter, we observed that (1) a long spacer provided enhanced antigen-dependent activation across all anti-GPRC5D CARs; and (2) the use of different scFvs resulted in vastly different levels of tonic signaling. We selected potential lead and backup constructs and evaluated CAR activity on primary human T cells. When co-cultured specifically with MM cell lines, anti-GPRC5D CAR T cells secreted a polyfunctional cytokine profile, proliferated, and effectively lysed target cells. CD138+/CD38hi primary MM bone marrow aspirate cells were also specifically lysed. In vivo efficacy of GPRC5D-targeted CAR T cells was evaluated in NSG mice engrafted with a human MM cell line (ffLuc+) bone marrow xenograft. Donor T cells were gene-modified to express anti-GPRC5D CARs with either a 4-1BB or a CD28 co-stimulatory domain and membrane-anchored Gaussia luciferase (GLuc). Compared to control CAR T cells specific for an irrelevant target, anti-GPRC5D CAR T cells with either co-stimulatory domain proliferated and homed to the site of MM (Gluc imaging), eradicated MM xenograft (ffLuc imaging), and increased survival (Figure 2). One scFv that was highly functional in our GPRC5D CAR screen was evaluated for off-target binding against either >200 G protein-coupled receptors (cell based), or >4000 human transmembrane proteins (scFv-Fc based), and demonstrated binding only to GPRC5D. Studies with murine and cynomolgus cross-reactive GPRC5D targeting CARs did not show signs of alopecia or other unexpected toxicity in either species. In a murine model of post-BCMA CAR T cell treated antigen escape (CRISPR BCMA KO of a subpopulation of MM cells), anti-GPRC5D CAR T cells rescue BCMA- relapse. These results indicate that GPRC5D will be an important target for the immunotherapy of MM. We are translating this 4-1BB-containing, human-derived, GPRC5D-targeted CAR construct to the clinic. Disclosures Smith: Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Harrington:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Masakayan:Agentus Inc: Employment. Jones:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Long:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Ghoddusi:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Pham:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Wang:Eureka Therapeutics: Employment, Equity Ownership. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Xu:Eureka Therapeutics: Employment, Equity Ownership. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Sather:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

We have developed a dual bioluminescent reporter system allowing noninvasive, concomitant imaging of T-cell trafficking, expansion, and activation of nuclear factor of activated T cells (NFAT) in vivo. NFAT activation plays an important role in T-cell activation and T-cell development. Therefore we used this system to determine spatial-temporal activation patterns of (1) proliferating T lymphocytes during graft-versus-host disease (GVHD) and (2) T-cell precursors during T-cell development after allogeneic hematopoietic stem cell transplantation (HSCT). In the first days after HSCT, donor T cells migrated to the peripheral lymph nodes and the intestines, whereas the NFAT activation was dominant in the intestines, suggesting an important role for the intestines in the early stages of alloactivation during development of GVHD. After adoptive transfer of in vitro-derived T-cell receptor (TCR) H-Y transgenic T-cell precursors into B6 (H-2b) hosts of both sexes, NFAT signaling and development into CD4+ or CD8+ single-positive cells could only be detected in the thymus of female recipients indicating either absence of positive selection or prompt depletion of double-positive thymocytes in the male recipients. Because NFAT plays an important role in a wide range of cell types, our system could provide new insights into a variety of biologic processes.

Patients with relapsed/refractory MM (RRMM) rarely obtain durable remissions with available therapies. Clinical use of BCMA targeted CAR T cell therapy was first reported in 12/2015 for RRMM, and based on small numbers, preliminary results appear promising. Given that host immune anti-murine CAR responses have limited the efficacy of repeat dosing (Turtle C. Sci Trans Med 2016), our goal was to develop a human BCMA targeted CAR T cell vector for clinical translation. We screened a human B cell derived scFv phage display library containing 6x1010 scFvs with BCMA expressing NIH 3T3 cells, and validated results on human MM cell lines. 57 unique and diverse BCMA specific scFvs were identified containing light and heavy chain CDR's each covering 6 subfamilies, with HCDR3 length ranges from 5-18 amino acids. 17 scFvs met stringent specificity criteria, and a diverse set was cloned into CAR vectors with either a CD28 or a 4-1BB co-stimulatory domain. Donor T cells transduced with BCMA targeted CAR vectors that conveyed particularly desirable properties over multiple in vitro assays, including: cytotoxicity on human MM cell lines at low E:T ratios (>90% lysis, 1:1, 16h), robust proliferation after repeat antigen stimulation (up to 700 fold, stimulation q3-4d for 14d), and active cytokine profiling, were selected for in vivo studies using a marrow predominant human MM cell line model in NSG mice. A single IV injection of CAR T cells, either early (4d) or late (21d) after MM engraftment was evaluated. In both cases survival was increased when treated with BCMA targeted CAR T cells vs CD19 targeted CAR T cells (median OS at 60d NR vs 35d p10,000 fold) CAR T expansion at day 6, followed by contraction of CAR T cells after MM clearance, confirming the efficacy of the anti-BCMA scFv/4-1BB containing construct. Co-culture with primary cells from a range of normal tissues did not activate CAR T cells as noted by a lack of IFN release. Co-culture of 293 cells expressing this scFv with those expressing a library of other TNFRSF or Ig receptor members demonstrated specific binding to BCMA. GLP toxicity studies in mice showed no unexpected adverse events. We generated a retroviral construct for clinical use including a truncated epithelial growth factor receptor (EGFRt) elimination gene: EGFRt/hBCMA-41BBz. Clinical investigation of this construct is underway in a dose escalation, single institution trial. Enrollment is completed on 2/4 planned dose levels (DL). On DL1 pts received cyclophosphamide conditioning (3g/m2 x1) and 72x106 mean CAR+ T cells. On DL2 pts received lower dose cyclophosphamide/fludarabine (300/30 mg/m2 x3) and 137x106 mean CAR+ T cells. All pts screened for BCMA expression by IHC were eligible. High risk cytogenetics were present in 4/6 pts. Median prior lines of therapy was 7; all pts had IMiD, PI, high dose melphalan, and CD38 directed therapies. With a data cut off of 7/20/17, 6 pts are evaluable for safety. There were no DLT's. At DL1, grade 1 CRS, not requiring intervention, occurred in 1/3 pts. At DL2, grade 1/2 CRS occurred in 2/3 pts; both received IL6R directed Tocilizumab (Toci) with near immediate resolution. In these 2 pts time to onset of fever was a mean 2d, Tmax was 39.4-41.1 C, peak CRP was 25-27mg/dl, peak IL6 level pre and post Toci were 558-632 and 3375-9071 pg/ml, respectively. Additional serum cytokines increased >10 fold from baseline in both pts include: IFNg, GM CSF, Fractalkine, IL5, IL8, and IP10. Increases in ferritin were limited, and there were no cases of hypofibrinogenemia. There were no grade 3-5 CRS and no neurotoxicities or cerebral edema. No pts received steroids or Cetuximab. Median time to count recovery after neutropenia was 10d (range 6-15d). Objective responses by IMWG criteria after a single dose of CAR T cells were observed across both DLs. At DL1, of 3 pts, responses were 1 VGPR, 1 SD, and 1 pt treated with baseline Mspike 0.46, thus not evaluable by IMWG criteria, had >50% reduction in Mspike, and normalization of K/L ratio. At DL2, 2/2 pts had objective responses with 1 PR and 1 VGPR (baseline 95% marrow involvement); 1 pt is too early to evaluate. As we are employing a human CAR, the study was designed to allow for an optional second dose in pts that do not reach CR. We have treated 2 pts with a second dose, and longer follow up data is pending. Figure 1 Figure 1. Disclosures Smith: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: BCMA targeted CAR T cells, Research Funding. Almo: Cue Biopharma: Other: Founder, head of SABequity holder; Institute for Protein Innovation: Consultancy; AKIN GUMP STRAUSS HAUER & FELD LLP: Consultancy. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xu: Eureka Therapeutics, Inc: Employment, Equity Ownership. Park: Amgen: Consultancy. Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Dogan: Celgene: Consultancy; Peer Review Institute: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

T cell therapies have had valuable clinical responses in patients with cancer. Chimeric antigen receptor (CAR) T cells can be genetically engineered to recognize tumor cells and CAR T cell therapy has shown impressive results in the setting of B cell acute lymphoblastic leukemia but has been less effective in treating other types of hematologic and solid tumors. The inhibitory tumor microenvironment (TME), including expression of ligands that bind inhibitory receptors on T cells, e.g. programmed death receptor 1 (PD-1), can dampen CAR T cell responses. Separately, immune checkpoint blockade therapy involving the disruption of PD-1 and programmed death receptor ligand1 (PD-L1) interaction allows for re-activation of tumor-infiltrating lymphocytes (TIL) to have anti-tumor function. This approach has shown clinical responses in a range of malignancies, but has been less efficacious in poorly immunogenic tumors. To prevent PD-1-mediated dampening of CAR T cell function, we have co-modified CAR T cells to secrete PD-1 blocking single chain variable fragments (scFv). We first designed mouse constructs with which we could investigate the scFv-secreting CAR T cells in the context of a syngeneic immune-competent intact TME. CAR constructs were engineered directed against either human CD19 or MUC-16 (ecto) with mouse signaling domains and a anti-mouse PD-1 scFv. Mouse T cells transduced with these constructs expressed the CAR on the surface and secreted detectable amounts of scFv that bound to mouse PD-1. The scFv-secreting CAR T cells were cytotoxic and produced IFN-g when co-cultured with PD-L1 expressing tumors in vitro . We utilized a syngeneic mouse model to study scFv secreting CAR T cells in a model with an intact TME. In tumor-bearing mice treated with CAR T cells, scFv-secreting CAR T cells enhanced survival as compared to second generation CAR T cells. The survival benefit achieved with scFv-secreting CAR T cells was comparable to that achieved with systemic infusion of PD-1 blocking antibody, but with localized delivery of PD-1 blockade. Mice treated with scFv-secreting CAR T cells had detectable scFv in vivo in the TME. Lastly, long term surviving mice had detectable CAR T cells in the bone marrow by PCR, demonstrating persistence and suggesting an immunological memory. We next aimed to translate PD-1 blocking scFv CAR T cells to a clinically relevant human model utilizing a novel anti-human PD-1 blocking scFv. CAR constructs were engineered with recognition domains directed against human CD19 or MUC-16 (ecto) and human signaling domains. Human T cells modified with the CAR constructs express the CAR on the surface and secrete detectable amounts of PD-1 blocking scFv. The scFv binds to human PD-1 and scFv-secreting CAR T cells are cytotoxic to PD-L1 expressing tumors. Expression of PD-1-blocking scFv enhances CAR T cell function against PD-L1 expressing tumors in xenograft models of hematological and solid tumors by enhancing survival in tumor-bearing mice as compared to second generation CAR T cells. Furthermore, scFv-secreting CAR T cells exhibit in vivo bystander T cell enhancement of function, suggesting scFv-secreting CAR T cells can reactivate endogenous TILs in the TME. These data support the novel concept that localized delivery of scFv by CAR T cells can successfully block PD-1 binding to PD-L1 and work in an autocrine manner to prevent dampening of CAR T cell responses as well as a paracrine manner to activate endogenous tumor infiltrating lymphocytes to enhance the overall anti-tumor efficacy of CAR T cell therapy. Disclosures Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Yan: Eureka Therapeutics Inc: Employment. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xiang: Eureka Therapeutics Inc.: Employment. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Chronic lymphocytic leukemia (CLL) is a hematological malignancy of B cells that accounts for a third of all new leukemia cases, but is incurable with standard first-line chemotherapies. Hematopoietic stem cell transplants offer the only curative treatment but are restricted to those with a matching donor. The recent advent of adoptive transfer therapy utilizing Chimeric Antigen Receptor (CAR) T cells, which are synthetic receptors that combine the target-specificity of antibodies to the effector capabilities of T cells, have emerged as a promising anticancer therapy due to its targeted tumoricidal activity. Clinical trials utilizing CD19-targeted CAR T cells have demonstrated strong efficacy against acute B-cell leukemia, but those performed in CLL have not been as successful for unclear reasons. This may be due to the dysfunctional tumor microenvironment which renders key immune effectors ineffective. Dendritic cells (DCs), a potent antigen presenting cell that functions as a bridge between the innate and adaptive immune systems, are rendered functionally and quantitatively deranged with an impaired ability to present antigen and thereby stimulate immunogenic responses. These limitations can potentially be rectified through an effective recruitment of an endogenous antitumor immune response and thus we hypothesize that genetically modified DCs should be capable of repolarizing the tolerogenic microenvironment induced by CLL toward one of immunogenicity to promote tumor eradication. In this study, we developed a qPCR assay to assess the DC CAR transcriptome. Because our CD19-targeted DC CARs with a CD40 intracellular domain signal via NF-κB to activate downstream elements, we needed to first assess the fidelity of our assay. Using the immortalized mouse DC cell line JAWS II, which lacks CD40, we used a potent TLR4 agonist, MPL, to stimulate our DCs through MyD88, an alternative pathway that also activates NF-κB. We saw elevated expression of downstream NF-κB elements, including IL-6, IL-10, IRF3, and IRF7. Establishing the sensitivity of our assay, we next evaluated the transcriptome of DC CARs by coculturing our CD19-targeted DC CARs containing a CD40 intracellular domain with HEK 293 cells, a human embryonic kidney cell line, retrovirally transduced with a human CD19 antigen to activate our CAR DCs and assess downstream gene expression. We observed expected elevation of downstream NF-κB elements including IL-6, IL-10, NOS2, IRF3, IFN-γ, TNFα, IL12p40, and TGF-β, indicating the viability of this assay. These positive results confirm the multidisciplinary potential of our assay as a promising technology that will allow us to better augment future CARs. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Therapies for acute myeloid leukemia (AML) have not improved patient long-term survival for decades. Novel treatment options, such as chimeric antigen receptor (CAR) T cells, are needed for patients with this disease. However, AML cells lack ideal targeting antigens that are safe to target with CAR T cells. CD33, a commonly targeted antigen, is expressed in about 85-90% of AML cases but is also present on normal myeloid progenitors and myelocytes. Our aim was to engineer and validate CD33-directed CAR T cells, with the intention to open a phase I clinical trial in patients with relapsed AML. Given the potential toxicity associated with targeting CD33 in patients, an elimination gene was included in the construct design to allow CAR T cell clearance after disease eradication. We constructed a CAR specific for CD33 by utilizing the variable heavy and light chains of the humanized M-195 antibody (HuM-195), CD28 and zeta signaling domains, and the IL-12 gene. To facilitate CAR T cell elimination if necessary, a truncated epidermal growth factor receptor (EGFRt) gene was also inserted into the retroviral vector to create the EGFRt/HuM195-28z/IL-12 CAR construct. Retroviral transduction with this tri-cistronic construct resulted in high transduction efficiency of human T cells, as determined by detection of EGFRt with fluorescently-labeled cetuximab or CAR with fluorescently-labeled CD33 molecule by flow cytometry. T cells transduced with the EGFRt/HuM195-28z/IL-12 CAR secreted functional IL-12, as assessed by culturing CAR cell supernatant with peripheral blood mononuclear cells and detecting IFN-g produced in response to IL-12. When stimulated through the CAR by co-culturing with the CD33+ AML cell line Molm-13, EGFRt/HuM195-28z/IL-12 CAR T cells proliferated and produced significant levels of the pro-inflammatory cytokines IFN-g and IL-2. In addition, EGFRt/HuM195-28z/IL-12 CAR T cells mediated significantly cytotoxicity against Molm-13 at a range of effector-to-target ratios in standard 51Cr-release assays, as compared to control CAR T cells. The in vivo anti-tumor efficacy of EGFRt/HuM195-28z/IL-12 CAR T cells was tested in two preclinical mouse models of AML. First, SCID/Beige mice were xenografted with Molm-13 cells. Mice subsequently treated with EGFRt/HuM195-28z/IL-12 CAR T cells exhibited significant long-term survival, as compared to untreated or control CAR T cell-treated mice (p = Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: CD19-targeted chimeric antigen receptor-modified (CAR) T cells have demonstrated considerable therapeutic efficacy in patients (pts) with relapsed and/or refractory (R/R) B cell ALL (B-ALL), resulting in rapid and often durable complete responses (CR). In contrast, a smaller subset of pts with R/R CLL have achieved CR following CD19-targeted CAR T cell therapy. Ibrutinib (IBR), which has considerable efficacy as a single agent in pts with R/R CLL, may modulate antitumor T cell immune responses. Others have observed enhanced ex vivo expansion of autologous T cells collected from pts with IBR exposure in response to CD3/CD28 bead stimulation, and improved CD19-targeted CAR T cell engraftment and antitumor efficacy in human xenograft models (Fraietta et al., Blood, 2016). Herein, we report on adults with CLL treated with IBR at the time of autologous T cell collection and/or around the time of CAR T cell infusion enrolled in our phase I clinical trial of CD19-targeted CAR T cells for adults with R/R CLL or B-cell NHL (NCT00466531). Methods: Eligible pts underwent leukapheresis and T cells were transduced with a retroviral vector encoding a CAR comprising a CD19-specific scFv and CD28 and CD3ζ signaling domains (19-28z). The present analysis is limited to pts with CLL. We identified pts with CLL treated with IBR at the time of leukapheresis and/or around the time of conditioning chemotherapy (CCT) and CAR T cell infusion. As a control group, we additionally identified all evaluable IBR-naïve pts with CLL treated on this study. Response was assessed by NCI-WG criteria. Cytokine levels were measured prospectively before and after CCT and CAR T cell infusion. Results: 5 pts (male, n=3), median age 58 at CAR T cell infusion (range, 43-66) with R/R CLL (TP53 loss, n=2) underwent therapy with IBR at leukapheresis (n=4) and/or immediately prior to or through CCT (cyclophosphamide [Cy], n=2; fludarabine [Flu]+Cy, n=3) and CAR T cell infusion (n=5). 6 additional evaluable pts with R/R CLL remained IBR-naïve through CCT (Cy, n=4; bendamustine, n=2) and CAR T cell infusion. A non-significant trend toward greater median cumulative fold T cell expansion ex vivo was noted in the 4 pts on IBR (vs the 7 not on IBR) at leukapheresis (374 [171-1518] vs 160 [49-468], p=0.13), with similar median manufacturing time (13.5 vs 15 days). End of process (EOP) T cells in pts undergoing collection while on IBR (vs those not on IBR) demonstrated a greater fraction of CD8+CAR+ T cells with a CD62L+CD127+ (central memory) phenotype (mean 29.0 vs 4.3%, p=0.10) and decreased fraction of CD62L- T cells (effector/effector memory phenotype) across CD8+CAR+ (mean 26.5 vs 54.4%, p=0.06) and CD4+CAR+ (mean 24.0 vs 57.8%, p=0.03) T cell subsets (Fig 1). IBR-treated pts received median 1x107 19-28z+ CAR T cells/kg (3x106-3x107/kg) and IBR-naïve pts received median 1x107 19-28z+ CAR T cells/kg (6x106-4x107/kg). Fevers developed in all 11 pts and began on the first day of infusion in 4/5 IBR-treated pts (vs 2/6 IBR-naïve pts); 2/5 IBR-treated pts (vs 0/6 IBR- naïve pts) developed severe CRS and required vasopressors for hypotension in addition to tocilizumab. IBR-treated pts additionally exhibited greater median peak levels of multiple immunoregulatory cytokines associated with CRS, including IL-6, IL-10, IL-2, IL-5, IFN-γ, FLT3L, fractalkine, and GM-CSF. In total, 5 of 11 enrolled pts with CLL (45%) treated with CCT and 19-28z CAR T cells achieved objective response (minimal residual disease [MRD]- CR, n=2; maintenance of MRD+ CR, n=1; PR, n=2); ORR was 4/5 among IBR-treated pts (1 MRD- CR, 1 MRD+ CR, 2 PR; p=0.08 for ORR between IBR-treated vs IBR-naïve pts). 2 pts remain in MRD- CR at 16 and 50 months. Maximal CAR T cell persistence observed to date is 159 days; peak vector copy levels by qPCR were highest in the 2 pts attaining MRD-negative CR. Conclusions: Prior therapy with IBR may influence EOP CAR T cell phenotypes. Prior ± concurrent IBR may improve antitumor responses following 19-28z CAR T cell administration, though small numbers of pts and differences in CCT regimens limit firm conclusions based on these data. Additionally, prior ± concurrent IBR may amplify CRS, though more intensive CCT (e.g. Flu/Cy vs Cy) may also enhance CAR T cell expansion in vivo and intensify CRS. Further strategies to overcome the inhibitory microenvironment and enhance CAR T cell expansion and efficacy in pts with R/R CLL are in preparation. Disclosures Park: Amgen: Consultancy; Genentech/Roche: Research Funding; Juno Therapeutics: Consultancy, Research Funding. Riviere:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed B-ALL. The primary objective of this study (NCT01860937) is to extend the use and test the safety of CD19 specific CAR T cells in a multicenter trial for children and young adults with relapsed CD19+ B-ALL. To date, 24 patients with very high risk (VHR) or relapsed B-ALL have been enrolled on protocol with a median age of 12 years (range 2-20 years) at time of T cell collection. We have treated 9 patients with relapsed B-ALL with a median age 15 years (range 3-22 years) using patient derived T cells expressing a CD19 specific CAR (19-28z). Patients received a dose of 1-3 x 10^6 CAR T cells/kg and complete response (complete remission or complete remission with incomplete count recovery) occurred in 5/9 (55%) patients. Significantly, correlations with response included lower disease burden (as assessed by bone marrow cellularity; p150 vs 20 fold), fractalkine (>20 fold), Flt-3L (>55 fold), IL-5 (>15 fold), IL-6 (>100 fold), and IL-10 (>15 fold) were demonstrated in patients with CRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T cell detection within 1-2 weeks following infusion with gradual contracture over 1-2 months. These early results demonstrate the feasibility and significant clinical impact of this approach in patients with relapsed B-ALL. In an effort to more rapidly generate statistically relevant data, demonstrate the "exportability" of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL we have expanded this trial to include a collaborating institution. The objective of our trial is not to provide an intent-to-treat cohort, but rather demonstrate the tolerability of this technology in patients with relapsed B-ALL. Furthermore, patients meeting disease eligibility were not pre-screened for lymphocyte function prior to collection and/or treatment. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. Disclosures Curran: Juno Therapeutics: Consultancy. Off Label Use: CAR T cells for B-ALL. Riviere:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Prockop:Atara Biotherapeutics: Other: I have no financial disclosures, but Atara Biotherapeutics has exercised a licensing agreement with Memorial Sloan Kettering Cancer Center and MSKCC and some investigators at MSKCC have a financial interest in Atara.. Park:Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy. O'Reilly:Atara Biotherapeutics: Research Funding. Sadelain:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Brentjens:Juno Therapeutics: Other: Co-founder, stockholder and consultant.

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

Cellular immune responses have the potential to elicit dramatic and sustained clinical remissions in lymphoma patients. Recent clinical trial data demonstrate that modification of T cells with chimeric antigen receptors (CARs) is a promising strategy. T cells containing CARs with costimulatory domains exhibit improved activity against tumors. We conducted a pilot clinical trial testing a “third-generation” CD20-specific CAR with CD28 and 4-1BB costimulatory domains in patients with relapsed indolent B-cell and mantle cell lymphomas. Four patients were enrolled, and 3 received T-cell infusions after cyclophosphamide lymphodepletion. Treatment was well tolerated, although one patient developed transient infusional symptoms. Two patients without evaluable disease remained progression-free for 12 and 24 months. The third patient had an objective partial remission and relapsed at 12 months after infusions. Modified T cells were detected by quantitative PCR at tumor sites and up to 1 year in peripheral blood, albeit at low levels. No evidence of host immune responses against infused cells was detected. In conclusion, adoptive immunotherapy with CD20-specific T cells was well tolerated and was associated with antitumor activity. We will pursue alternative gene transfer technologies and culture conditions in future studies to improve CAR expression and cell production efficiency. This study is registered at www.clinicaltrials.gov as NCT00621452.

Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens. Disclosures Dao: Novartis: Patents & Royalties. Liu:Eureka: Employment, Inventor Other. Scheinberg:Novartis: Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed B-ALL. The primary objective of this study (NCT01860937) is to extend the use and test the safety of CD19 specific CAR T cells in children with relapsed CD19+ B-ALL. To date, 11 pediatric patients with very high risk (VHR) or relapsed B-ALL have been enrolled on protocol with a median age of 13 years (range 2-23 years) at time of T cell collection. We have treated 4 pediatric patients with relapsed B-ALL (ages 13, 14, 19, and 22 years) using patient derived T cells expressing a CD19 specific CAR (19-28z). Complete response (complete remission or complete remission with incomplete count recovery) occurred in 2/4 (50%) patients. Severe cytokine release syndrome (sCRS) defined by fever for ≥3 consecutive days, elevated serum cytokine levels, and one clinical sign of toxicity (hypotension, hypoxia, neurologic disorder including altered mental status, obtundation, and/or seizure) occurred in both patients who responded to CAR T cells. Morphologic disease (≥5% bone marrow blasts) at time of treatment was present in three patients including both patients with sCRS. Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of sCRS. Elevated serum cytokines of IFN-g (>20 fold), fractalkine (>20 fold), Flt-3L (>55 fold), IL-5 (>15 fold), IL-6 (>100 fold), and IL-10 (>15 fold) were demonstrated in patients with sCRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T-cell detection within 1-2 weeks following infusion with gradual contracture over 2-3 months. These early results demonstrate the feasibility and significant clinical impact of this approach in patients with relapsed B-ALL. To more rapidly generate statistically relevant data, demonstrate the “exportability” of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL our trial will expand into a phase I multicenter clinical trial with a collaborating institution. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. Disclosures Off Label Use: CAR T cells for relapsed B-ALL. Riviere:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Boulad:Genzyme Sanofi: Trials partially funded by Genzyme Sanofi Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Background: Relapsed adult acute lymphoblastic leukemia (ALL) is associated with high reinduction mortality, chemotherapy resistance, and dismal prognosis with a median overall survival (OS) < 6 months and 5-year OS ≤10%. We have previously reported a high anti-tumor activity of autologous T cells genetically modified to express 19-28z chimeric antigen receptor (19-28z CAR) targeting CD19 in adult patients with CLL and ALL (Brentjens R et al. Blood 2011; Davila M et al. Sci Transl Med2014). Herein, for the first time, we further report the long-term outcome of our phase I clinical trial in adults with relapsed/refractory (R/R) ALL (NCT01044069) with analysis on potential predictive markers of response and neurological toxicities. Patients and Methods: Adult patients with R/R B-ALL were enrolled. Eligible patients underwent leukapheresis, and T cells were transduced with a retrovirus encoding a CAR construct composed of anti-CD19 scFV linked to CD28 and CD3ζ signaling domains (19-28z). All patients received lymphodepleting chemotherapy followed 2 days later by 1x106 – 3x10619-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells in ALL. Post-treatment minimal residual disease (MRD) was assessed at day 14-28 by multiparameter flow cytometry and deep sequencing in the bone marrow (BM) samples (Adaptive Biotech Corp.) Results: 24 patients have been treated. The median age was 56 years (range, 23-74). 6 patients (25%) had Ph+ B-ALL (T315I mutation in 2 patients), 6 patients (25%) had prior allogeneic hematopoietic stem cell transplant (allo-HSCT), and 11 patients (46%) had 3 or more prior lines of ALL therapy before receiving the 19-28z CAR T cell therapy. Of the 24 patients, 22 patients were evaluable for response. At the time of 19-28z CAR T cell infusion, 12 of 22 patients had morphologic disease (6 to 97% blasts in the BM) and the remaining 10 patients had MRD. Twenty out of 22 patients (91%) were in complete remission (CR) after 19-28z CAR T-cell infusion, and 18 of these 20 patients (90%) achieved an MRD-negative CR. Ten of the 13 transplant eligible patients (77%) successfully underwent allo-HSCT following the 19-28z CAR T cell therapy. As of July 1, 2014, the median follow-up was 7.4 months (range 1-34), with 13 patients having at least 6 months of follow-up. Responses appear durable with 6 patients remaining disease-free beyond 1 year (range 12.6 – 34 months). Median overall survial (OS) is 9 months. 5 patients relapsed during the follow-up, including 1 patient with CD19 negative relapse. Three of the relapsed patients were treated again with the 19-28z CAR T cells, and two patients achieved a second CR. Comparing responders to non-responders, no association was observed between response and age ( While none of the 10 patients with MRD at the time of T cell infusion developed cytokine release syndrome (CRS), 9 of 13 patients with morphologic disease at the time of the T cell infusion developed CRS with or without neurological symptoms that required intervention with an IL-6R antagonist or corticosteroid. A detailed analysis of serum cytokines demonstrated a consistent peak of IL-6 (22.2 to 553-fold increase) immediately prior to the development of neurological toxicities. Based on these data, we have developed a multi-disciplinary CRS management algorithm for patients at high risk in order to reduce the severity of CRS and improve safety of the 19-28z CAR T cell therapy. Conclusions: While longer follow-up is needed to confirm the durability of the observed responses, the potent induction of MRD-negative responses and successful long-term outcomes, including subsequent allo-HSCT without apparent additional post-transplant toxicities, strongly support the use of 19-28z CAR T cells in adult patients with B-ALL. A temporal relationship between serum IL-6 levels and neurological toxicities indicates that early intervention with IL-6 directed therapy may be more effective in ameliorating neurological toxicities in patients with morphologic disease at the time of T-cell infusion. These findings will need to be evaluated systematically and confirmed in a larger phase 2 trial. Disclosures Park: Juno Therapeutics: Research Funding. Riviere:Juno Therapeutics: Consultancy, scientific co-founders Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-NHL. While 50-80% of patients with DLBCL are cured with standard induction therapy, a large fraction of patients either relapse or have primary refractory (rel/ref) disease. High-dose therapy followed by autologous stem cell transplantation (HDT-ASCT) is the established standard of care for these patients. Despite this, approximately half of all rel/ref patients that are chemosensitive to salvage therapy are cured with this approach. Relapse is the major cause of treatment failure, and ultimately death, in these patients. Our group has demonstrated encouraging activity with 19-28z chimeric antigen receptor modified T cells (19-28z CAR-T) directed against CD19 in rel/ref ALL. We hypothesize that biologic optimization of cellular therapy with 19-28z CAR-T can be met immediately post-HDT-ASCT given: lymphoproliferative cytokine availability through lymphodepletion post-HDT, depletion of prohibitive regulatory cellular elements post-HDT and achievement of minimal residual disease prior to 19-28z CAR-T consolidation. Herein, we report safety data on the first 6 patients of the phase I MSKCC #12-117: 19-28z CAR-T post HDT-ASCT for poor-risk rel/ref aggressive B-NHL. Methods: Eligibility for this study includes rel/ref aggressive B-NHL appropriate for HDT-ASCT as defined by chemosensitivity to salvage therapy and poor risk features including: 1) FDG-PET positivity following 2 cycles of salvage therapy or 2) bone marrow involvement of B-NHL at the time of rel/ref clinical restaging. Patients underwent separate apheresis for CD34+ progenitor cells and CD3+ T cells. T cells were transduced with a retrovirus encoding a CAR construct composed of anti-CD19 scFV linked to CD28 and CD3ζ signaling domains (19-28z). Patients were admitted for BEAM conditioned HDT and ASCT occurred day 0. Pegfilgrastim was administered on day+1 and 19-28z CAR-T dose per phase I study was split on days +2 and +3. Results: This analysis includes the first six patients, all male, on the phase I study. The median age is 61 (range 34-68) years at the time of HDT-ASCT. Diagnoses included: n=2 relapsed and transformed follicular lymphoma (one with double-hit biology), n=3 relapsed DLBCL (one CD5+) and one subject with relapsed and transformed marginal zone lymphoma involving the bone marrow. Five patients were treated at dose level #1 (5 x106 19-28z CAR-T/kg) with no dose-limiting toxicity (DLT) observed. Four of the five patients at dose level #1 experienced grade 3 febrile neutropenia and one patient met-criterion for non-severe cytokine-release syndrome (nCRS) effectively treated with tocilizumab 4 mg/kg x1. One patient was treated at dose-level #2, 1 x107 CAR-T/kg, and experienced a DLT of grade 4 severe CRS (sCRS) manifested with acute kidney injury, hypotension and mental status changes effectively treated and fully recovered with tocilizumab in combination with dexamethasone. Peak CRP in all patients was observed at a median of 3.5 days (range 3-4 days) post-19-28z CAR-T infusion (median peak CRP= 17 mg/dL, range: 5-43.1 mg/dL), with CRP >20 mg/dL identified in the two patients that experienced CRS (nCRS=27 mg/dL, sCRS=43 mg/dL). Previously associated serum cytokine elevations (Davila et al Sci Trans Med, 2014) were observed in the two patients that experienced CRS (Figure). All patients engrafted neutrophils at a median of 10 days (range: 9-10 days) post-ASCT, and achieved a complete remission at first post-HDT-ASCT restaging. No sequelae of autoimmune phenomenon were observed. At a median follow-up of six months, with 2 patients >one year post HDT-ASCT, all patients remain alive and in remission. Conclusions: This is the first report of 19-28z CAR-T cells in conjunction with consolidative HDT-ASCT for poor-risk rel/ref aggressive B-NHL. No DLTs have been observed at dose level #1, 5 x106 19-28z CAR-T/kg, while sCRS was observed in n=1 at 1 x107 CAR-T/kg resulting in a DLT. CRS was associated with pro-inflammatory serum cytokine elevation. CRS was aborted with tocilizumab with (sCRS=1) or without (nCRS=1) dexamethasone. All patients engrafted neutrophils at the expected time point. The use of 19-28z CAR T cells is a promising approach in this small group of poor-risk PET+ NHL patients undergoing autologous transplant. This is an ongoing trial and updated data will be presented. Figure 1 Figure 1. Disclosures Riviere: Juno Therapeutics: Consultancy, scientific co-founders Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, scientific co-founder Other.

Chronic Myeloid Leukemia (CML) is a rare myeloid neoplasm with an age-adjusted incidence of 1.6 per 100,000 people in the United States (http://seer.cancer.gov/statfacts/html/cmyl.html). Despite the understanding of the pathogenesis of CML – the fusion of BCR-ABL leading to a constitutively activated tyrosine kinase – underlying events that lead to a 9;22 translocation are incompletely elucidated. Although CML was the most common leukemia seen among survivors of Hiroshima and Nagasaki, other causative factors are not well defined. There is a known association between exposure to radiation and/or cytotoxic chemotherapy and therapy-related acute myeloid leukemia (t-AML). This relationship is best described after adjuvant treatment for breast cancer (BC), but has also been reported in patients treated for lymphoma, testicular cancer and colorectal cancer. Based on a number of patients we have seen at Memorial Sloan-Kettering Cancer Center with CML who had prior adjuvant treatment for BC, we hypothesized that a subset of patients with CML have therapy-related disease and the acquisition of a 9;22 translocation may be related to prior exposure to cytotoxic chemotherapy and/or radiation. Using ICD-9 codes for BC, we queried the Memorial Sloan-Kettering institutional database from 1983-2012 to search for patients who had a co-occurring diagnosis of CML, as defined by a bone marrow aspirate or biopsy that was morphologically consistent with CML and either a cytogenetic report that confirmed a 9;22 translocation and/or a BCR-ABL p210 fusion transcript. Charts of patients identified in the institutional database were individually reviewed to confirm their eligibility for this retrospective study. In total, 15 patients who developed a diagnosis of CML after adjuvant treatment of BC were identified (table 1). Of those identified, all had received prior adjuvant chemotherapy (n=11), prior adjuvant radiation (n=12) or both (n=15). The cumulative doses of anthracycline and alkylating agents are detailed in table 1. The interval between the adjuvant treatment of breast cancer and CML was 4.7 years. Patients diagnosed with CML after treatment of BC tended to be younger than those with CML diagnosed in the general population (50.47 years vs. 64 years) and were overwhelmingly Caucasian. All patients were diagnosed in chronic phase and none of the patients had an additional cytogenetic abnormality. The cumulative incidence of CML was markedly higher than in the general population; while 78,000 unique patients with BC were seen at MSKCC between 1985 and 2012, 15 patients subsequently received a diagnosis of CML. This incidence is considerably greater than the age-adjusted incidence of 1.6 per 100,000 in the United States population.Table 1Patient CharacteristicsTotal15At time of Breast Cancer DiagnosisMedian Age (years)*46.59 (38-73)Race Caucasian13Asian2 African-American0Adjuvant Chemotherapy11 Anthracycline (n)5 Cumulative dose*240 mg/m2 Alkylator (n)10 Cumulative Dose*4800mg/m2Adjuvant Radiation12At time of CML DiagnosisMedian Age (years)*50.42Interval - Breast Ca and CML (years)*4.7 (1.2-18.8)White count at presentation*31 (24-188)Hgb at presentation*12.5 (7.9 – 14.1)Platelets at presentation*397 (185-1082)Initial Treatment Imatinib13 Dasatinib or Nilotinib0 Other2#*Median (range)#One patient received hydrea and one received an allogeneic stem cell transplant To our knowledge, our data represent the first comprehensive description of CML after adjuvant treatment of BC. The markedly elevated incidence of CML after receipt of adjuvant chemotherapy and/or radiation for BC as compared to the general US population suggests that this entity might be classified as a therapy-related leukemia. The exact pathogenesis of CML after adjuvant treatment for BC and reason patients more commonly develop t-AML requires further investigation. Given the remarkable efficacy of tyrosine kinase inhibitors in preventing progression to accelerated or blastic phase disease, the practicing oncologist focused in breast cancer should consider a diagnosis of t-CML in any BC patient, previously treated with adjuvant chemotherapy or radiation, who presents with an elevated white blood count.

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

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

Abstract 2613 Recent work by our group and others demonstrates the therapeutic potential of CD19-targeted T cells to treat patients with indolent B cell malignancies. These studies make use of T cells that are genetically engineered with chimeric antigen receptors (CARs) comprising an scFv fused to various T cell activating elements. Whereas firs-generation CARs only direct T cell activation, second-generation CARs include two signal elements, such as CD3z and CD28 signaling domains (19–28z). We and our colleagues at MSKCC are currently evaluating the safety of 19–28z-transduced T cells in patients with acute leukemia (B-ALL) in a Phase I protocol (NCT01044069). Pre-clinical studies performed to date have mostly relied on xenogeneic models utilizing immunodeficient animals, which enable the evaluation of human engineered T cells but do not recapitulate all the interactions that may affect tumor eradication by CAR-modified T cells. We have therefore developed a pre-clinical immunocompetent mouse model of B-ALL, and addressed therein the impact of conditioning and T cell dose on the eradication of leukemia by syngeneic, CAR-targeted T cells. To establish an immunocompetent mouse model of B cell leukemia, we generated a clone from the lymph node of an Eμ-myc B6 transgenic mouse. The immunophenotype and gene-expression profile of clone Eμ-ALL01 is consistent with a progenitor B cell origin. Syngeneic B6 mice inoculated with this clone develop florid acute leukemia and die approximately 2–4 weeks after injection from progressive bone marrow infiltration. We created an anti-mouse CD19 CAR comprising all murine elements, including the CD8 signal peptide, a CD19-specific single chain variable fragment, the CD8 transmembrane region, and the CD28 and CD3z signaling domains. Transduction of the murine 19–28z CAR into mouse T cells was robust and successfully retargeted the T cells to B cells. In vitro assays demonstrated that m19–28 z transduced T cells mediated effective killing of CD19-expressing target cells and the production of effector cytokines such as IFNγ and TNFα. Cyclophosphamide either alone or in combination with control syngeneic T cells is insufficient to eradicate established Eμ-ALL01 in B6 mice. However, treatment with cyclophosphamide and m19–28z-transduced T cells cured nearly all mice. Mice sacrificed six months after treatment exhibited a dramatic reduction of B cells in the bone marrow (BM), blood, and spleen. The few remaining B lineage cells found in the BM had a phenotype consistent with early pro-B cells, suggesting that endogenous reconstitution of the B cell compartment was thwarted by persisting, functional m19–28z+ T cells. Thus, T cells are retained at the site of antigen expression, which is maintained through regeneration of progenitor B cells. The persisting CD19-targeted T cells in the BM exhibited a cell surface phenotype consistent with effector and central memory cells. Using B cell aplasia as a surrogate endpoint for assessing in vivo T cell function and persistence, we evaluated how conditioning chemotherapy and T cell dose determine the level of B cell depletion induced by adoptively transferred CD19-targeted T cells. Overall, increasing the cyclophosphamide or T cell dose, increased the degree and duration of B cell depletion and the number of persisting CAR-modified T cells. Significantly, increasing the T cell dose at a set cyclophosphamide level had a lesser impact than increasing the conditioning intensity for a given T cell dose. In summary, the new Eμ-ALL01 syngeneic, immunocompetent B-ALL model we describe here is a valuable tool for modeling CD19 CAR therapies. Our results indicate that m19–28z transduced T cells are effective at eradicating B-ALL tumor cells and persist long-term, preferentially in bone marrow. Our findings further establish that conditioning intensity and T cell dose directly determine B cell elimination and long-term T cell persistence. These studies in mice will serve as an important framework to further model and perfect our studies in patients with B-ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 353 T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR modified T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapse B-ALL following allo-HSCT. We have previously demonstrated donor derived Epstein-Barr virus specific cytotoxic T cells (EBV-CTLs) can be safely infused in patients following allo-HSCT. Following retroviral gene transfer with our CD19-specific CAR (19–28z), EBV-CTLs demonstrate in vitro cytotoxicity against CD19+ targets, retain EBV-specificity without allogeneic reactivity, and in vivo, eradicate systemic Burkitt lymphoma in a SCID/Beige murine model. Based on our preclinical studies we have initiated a phase I dose escalation clinical trial (NCT01430390) in pediatric patients with relapsed CD19+ B-ALL following allo-HSCT utilizing donor 19–28z+ EBV-CTLs. Three patients have received conditioning chemotherapy (fludarabine 25mg/m2 × 5 days) followed by infusion of CAR modified EBV-CTLs without infusion related toxicity. Subsequently, patient 1 and 3 safely received additional T cell infusions without (patient 1) and with (patient 3; cyclophosphamide) conditioning chemotherapy. Total CAR+ T cell dose for each infusion ranged from 1.4 – 4.1×105 cells/kg (1×106 total EBV-CTLs/kg per infusion). Patient 1 developed fever and biopsy proven skin GVHD (grade II) three days following his first infusion which resolved 2 weeks after starting topical steroids. Modified T cells were detected by Q-PCR in the bone marrow and peripheral blood up to 3 and 18 weeks respectively in patient 1 following first infusion, up to 2 weeks in the peripheral blood in patient 2, and have been below the limit of detection in patient 3. Patient 1 died from progressive disease 29 weeks following first infusion and patient 2 died from progressive disease 19 weeks post infusion. Patient 3, initially treated in remission following salvage chemotherapy, has no evidence of disease (NED) 15 months after last extramedullary relapse and 5 months post first infusion of 19–28z+ EBV-CTLs. These early results demonstrate the feasibility of allogeneic 1928z+ EBV-CTL infusions without the development of significant GVHD post allo-HSCT. Subsequent cohorts of patients will receive dose escalation of modified T cells and will be evaluated for safety, persistence of 19–28z+ EBV-CTLs, and for anti-tumor efficacy. Disclosures: No relevant conflicts of interest to declare.

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

Abstract 2602 Novel therapeutic approaches require preclinical in vivo testing before first use in men. An important preclinical model to study acute lymphoblastic leukemia (ALL) was first described more than 2 decades ago (Dick et al., Science 1989). In this model, primary human tumor cells from patients with ALL are transplanted into severely immuno-compromised mice. The model is hampered by the lack of a sensitive readout for disease progression and treatment effects. As ALL is a systemic disease per se, tumor mass is difficult to measure; bone marrow aspirations remain exhausting for mice; secretion of tumor cells into the blood stream remains a late and only partially reliable surrogate parameter. Here, we optimized the preclinical individualized mouse model of ALL by introducing a highly sensitive and reliable readout parameter for disease progression and treatment efficacy. We introduced the lentiviral transduction of patient-derived ALL cells to express transgenes such as luciferase and fluochromes. Transduction efficiency ranged between 0,5 and 70 %, depending on the sample used. Expression of fluochromes was used for enrichment of transgenic cells by FACs sorting. Expression of luciferase was used to perform bioluminescence in vivo imaging in mice. Bioluminescence in vivo imaging was easy to perform and well tolerated by the mice, even as often as 3 times per week. Imaging enabled highly reliable measurement of disease progression over time in single mice and revealed a strictly logarithmic growth of leukemia in mice. Imaging results correlated perfectly well with post mortem determination of leukemic burden using immunohistochemistry and FACs analysis. In contrast to the later, imaging was highly sensitive and detected leukemia at disease stages representing minimal residual disease. Depending on the expression level of the transgenes, imaging was able to detect as few as 1 human ALL cells in >> 10.000 normal mouse bone marrow cells. By following up groups of mice over time, imaging revealed especially helpful for readout of limiting dilution transplantation assays to determine frequencies of leukemia initiating cells, e.g., with and without prior in vitro treatment. In preclinical treatment trials on ALL cells from individual patients, imaging quantified therapy effects precisely and with low variances discriminating treatment failure from partial and complete response as early as 4 days after treatment. Due to follow up of mice over time, imaging visualized disease regrowth after successful therapy and allowed determining classical clinical readout parameter such as time to tumor regrowth. Taken together, the most demanding challenges in anti-cancer treatment are modeled precisely and individually in mice using bioluminescence in vivo imaging. In vivo imaging upgrades the individualized xenograft mouse model of ALL by highly sensitive and reliable disease monitoring. Imaging enables performing preclinical trials at defined disease stages and using classical clinical readout parameters. Imaging-based individualized preclinical trials of ALL will facilitate translation of novel therapeutic approaches from bench to bedside. Disclosures: No relevant conflicts of interest to declare.

Abstract 2482 Despite the advent of prognostic factors such as cytogenetics and IGH mutational status, the natural history of chronic lymphocytic leukemia (CLL) remains heterogenous and difficult to predict in individual patients. We used the carbocyanine dye, JC-1, to investigate mitochondrial membrane potential in patients with untreated, predominantly early stage CLL. We found that JC-1 staining separated CD5+/CD19+ CLL cells into two discrete populations, Low Red (R)/Green (G) and High R/G, which mainly differed with regard to JC-1 green fluorescence (530 nm) in all patients (Fig. 1). In evaluating CLL patients, we found substantial variation in the proportion of cells in the two populations, with a range of 1.1% to 96.7% (median=79.9%) in the High R/G population. CD19+ B-cells from normal individuals typically had few cells with high JC-1 red fluorescence and had a JC-1 staining pattern that was distinct from CLL patients. Electron microscopy revealed disorganized mitochondrial christae, particularly in patients with elevated High R/G cell populations, compared with mononuclear cells from normal donors. Consistent with the morphologic disorganization, we found that the two populations of CLL cells had differential survival in culture with pyruvate and glucose as primary energy sources; cells in the High R/G population had inferior survival (p=0.016), while cells in the Low R/G population had equal survival in pyruvate compared with glucose (Fig. 2). This result indicates that the CLL clonal cell population can be subdivided into cells able to use oxidative phosphorylation and those dependent on aerobic glycolysis fro metabolic energy needs. Interestingly, patient samples with High R/G cell populations >90% had better cell survival in glucose-containing medium than samples derived from patients with < 75% High R/G cells (p=0.0085). We found that the mononuclear cells from patients with CLL expressed the M2 isoform of pyruvate kinase, which has been linked to the regulated utilization of aerobic glycolysis. We assessed the outcome of 100 patients with untreated CLL with more than 1 month of follow up (median 36 months) after measuring the proportion of cells in the mononuclear fraction of the peripheral blood with in the High R/G population. The clinical characteristics of the patients at the time of JC-1 staining are characteristic of patients with early stage, untreated CLL. The population was divided into approximate quartiles based on the percentage of mononuclear cells in the High R/G population, and progression-free survival (PFS) was estimated within each quartile. Patients with 50% or fewer cells in the High R/G gate appeared to have an indolent natural history; an estimated 88% (95% CI: 73%, 99%) of the patients had no evidence of significant clinical progression at two years. In contrast, patients with more than 90% of cells in the gate with High R/G cells had rapid progression with an estimated PFS of 50% (95% CI: 31%–82%) at two years (Fig. 3). Patients with 50–90% cells in the High R/G gate had an intermediate disease course. There was strong evidence of a difference in survival distributions across the four JC-1 categories (log-rank p-value < 0.001). The percentage of cells in the High R/G gate provided prognostic information that was independent of good risk or poor risk cytogenetic changes. In summary, we found that the clonal, neoplastic cells in patients with CLL comprise discrete populations separated on their metabolic dependence on aerobic glycolysis and aerobic glycolysis. We also show that the percentage of cells utilizing glycolysis has a significant adverse impact on the natural history of patients with CLL. Aerobic glycolysis, the so-called Warburg effect, is a common and incompletely understood aspect of cancer biology. These data tie the acquisition of aerobic glycolysis to more aggressive cancer biology, identify an important new prognostic measure for CLL and could lead to the development of therapies that target metabolic differences in cancer cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 4433 One of the aims of the National Comprehensive Cancer Network (NCCN) is to provide physicians with state-of- the- art treatment pathways for specific malignancies. These guidelines are frequently updated, and disease- based committee members either meet or speak with each other at least once a year. However, it is not known to what extent these guidelines are actually used in clinical practice. In order to determine how physicians in a single large academic cancer center utilize NCCN guidelines for patients with chronic myelogenous leukemia (CML), we reviewed 20 randomly chosen patients with CML diagnosed between 2002 –2007 and 2008– 2010, using 2008 as the year when the more contemporary testing schedule for FISH and PCR was established. Nine physicians, median age 51 years (range 35–61) with a median clinical care experience of 21 years (range 2–30), cared for these patients. The median age of the patients was 58 (range 22–56), 11 were male, and median follow up was 4 years (range 1–8). Eleven patients began treatment in 2008 or later. CML guidelines were divided into two main components: (1) documentation of CML by bone marrow (BM) studies at time of diagnosis, and after 6 and 12 months of imatinib (IM) therapy, and (2) frequency of testing with either BM or peripheral blood (PB) FISH and PCR for BCR-ABL. Of the 20 patients, 17 had diagnostic BMs performed on their initial visit here, and 3 had PB confirmation of disease, having had BMs done on the outside 1, 2, and 5 months prior to their initial visit. Thirteen of the 20 patients had BMs performed after 6 months of IM therapy, and 11 had BMs performed at 12 months; 5 patients had negative karyotype and FISH studies on their 6th month BM and thus did not have a repeat BM at month 12. Thus, NCCN guidelines were followed in 80% of patients. The most common deviation from NCCN guidelines was the frequency of interim PB and/or BM testing using FISH and PCR. While the median interval for PB testing was every 3 months (range 1 to 6), significant numbers of patients had much more frequent testing. For example, 13 of the 20 patients had serial PB FISH performed after at least one negative FISH result; 3 patients had > 5 samples analyzed and one patient had 10 samples tested after at least one negative result. In all these patients, subsequent FISH tests results after the first negative test were also negative. Two patients had BM testing performed after PB PCR was negative in at least two prior samples; BM PCR results were also negative. Three patients had simultaneous BM and PB PCR performed which provided similar results. At the time of last testing, 19 of the 20 patients had a complete cytogenetic response, 13 patients had a complete molecular response, and 6 patients had a major molecular response. One patient with significant co-morbidities took IM intermittently and had no response to therapy. There were no differences between the two groups with regards to frequency of BM or PB FISH or PCR testing. In summary, in this CML patient population, important therapeutic tests such as BM sampling at diagnosis and at treatment decision points (12 months) were met in the majority of patients. However, interim testing varied widely and in many instances, was redundant, even after publication of NCCN contemporary guidelines. In order to eliminate such unnecessary testing and conserve important resources, improved internal audits and communication between the laboratory and the clinical staff is needed. Disclosures: Jurcic: Actinium Pharmaceuticals, Inc.: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Lamanna:Celgene: Membership on an entity’s Board of Directors or advisory committees.

Abstract 3120 T cells may be genetically modified to target tumor-associated antigens through the retroviral transduction of genes encoding chimeric antigen receptors (CARs). We have previously generated a series of CARs specific to the CD19 antigen expressed on most B cell tumors. In xenotransplant models of disease we have shown that human T cells expressing CD19 targeted CARs successfully eradicate established B cell tumors in immune compromised SCID-Beige mice. To further our understanding of the in vivo biology of CAR modified T cells, we generated a more clinically relevant syngeneic, immune competent tumor model utilizing CD19 knock out (mCD19−/−) human CD19 knock in (hCD19+/−) C57BL6 transgenic mice (C57BL6(mCD19−/− hCD19+/−)) bearing systemic syngeneic EL4(hCD19+) thymoma tumors. Treatment of tumor bearing mice with syngeneic T cells modified to express the CD19 targeted 19m ζ CAR alone failed to either eradicate tumor or induce predicted B cell aplasias. However, prior lymphodepletion with cyclophosphamide followed by infusion of 19m ζ+ T cells successfully eradicated tumor in 83% of treated mice and induced long term B cell aplasias. Translation of this therapy to the clinic has also revealed that optimal responses require pre-conditioning regimens. Given the toxicity of pre-conditioning treatments, these therapies are largely restricted to younger, healthier patients able to tolerate such intensive regimens. To further understand the mechanisms of action of improved therapy following prior cyclophosphamide therapy, we demonstrated markedly enhanced serum levels of the IL-12 and IFN γ cytokines as well as a marked reduction of endogenous CD4+ regulatory T cells. We postulated that IL-12, which induces IFN γ secretion, may in part explain the enhanced anti-tumor efficacy following prior lymphodepletion, and may potentially obviate the need for toxic conditioning pre-treatments. To address this hypothesis, we modified CAR+ T cells to constitutively secrete IL-12. Subsequent adoptive therapy of EL4(hCD19+) tumor bearing C57BL6(mCD19−/− hCD19+/−) mice with hCD19 targeted IL-12 secreting T cells successfully eradicated disease in 75% of treated mice and induced predicted B cell aplasias in the absence of prior lymphodepletion. Significantly, we found that this enhanced hCD19 targeted T cell activity required the infusion of both CD4+ and CD8+ gene modified T cells, and was further dependent upon autocrine IL-12 stimulation of the modified tumor targeted T cells as well as modified T cell IFN γ secretion and stimulation. To our knowledge, this is the first time adoptive T cell transfer has been demonstrated to successfully eradicate tumor in the absence of prior conditioning regimens. Therefore, these data support the rapid translation of this adoptive T cell therapy to the clinic, as it may enhance the anti-tumor efficacy of this therapy and further expand the patient population eligible for adoptive T cells therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3121 Cellular therapies through allogeneic hematopoietic stem cell transplants (allo-HSCT) and infusion of genetically modified T cells targeted against tumor associated antigens hold great promise for cancer therapy. However, significant risk of graft-versus-host-disease (GvHD) with allo-HSCT and potential toxicities from the transgene products in the case of genetically modified T cells have generated considerable interest in developing safeguards in the form of suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events. We have previously reported the comparable in vitro function of 3 different suicide gene-prodrug combinations (Park et al. ASH 2010 abstract #3771). The three suicide genes examined included herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and a novel suicide gene nitroreductase (NTR) derived from E.coli combined with the prodrug metronidazole. Here, we report the comparison of in vivo efficacy of these 3 suicide gene-prodrug combinations using our previously published mouse GvHD model (Santos et al. Nat Med 2009). SCID-Beige mice were injected i.v. with 1×106 FACS-sorted major histocompatibility complex mismatched C57/BL6 T cells transduced to express each suicide gene (HSV-TK SR39, iCasp9 or NTR) cloned in frame with external Gaussia Luciferase (extGLuc), separated by the 2A peptide in an SFG-based vector. Seven days after intravenous injection of modified T cells, SCID-Beige mice developed GvHD as evidenced by in vivo expansion of the transferred T cells detected by bioluminescent imaging (BLI) after a bolus injection of coelenterazine. Subsequently, the mice injected with the HSV-TK SR39-transduced T cells were treated intraperitoneally (i.p.) twice daily for 5 days with ganciclovir at 25mg/kg; the mice with the NTR-transduced T cells were treated i.p. three times daily for 5 days with metronidazole at 200μg; the mice with the iCasp9-transduced T cells were treated i.p. daily for 3 days with CID at 50μg; and the mice with the HSV-TK SR39-transduced T cells treated i.p. twice daily for 5 days with normal saline served as a control group. All treated mice were imaged by BLI on days 3, 7, 11, 18, 25, 32, and weekly thereafter for a total of 3 months following the first dose of each prodrug. All of the control mice treated with normal saline developed rapidly progressing GvHD both clinically and by BLI, meeting the criteria for euthanasia by day 18. In contrast, the administration of prodrugs successfully eradicated the modified T cells expressing respective suicide genes by day 3 and no evidence of GvHD was detected. However, a long-term follow-up of these mice revealed the re-expansion of the transferred T cells or relapse of GvHD in all mice in the NTR group (by day 39) and in 2 out of 3 mice in the HSV-TK SR39 group (by day 53), but none in the mice in the iCasp9 group. The relapsed mice in the NTR and HSV-TK SR groups were treated again with the prodrugs, but none responded to the re-treatment. At the end of the 3 month follow-up, all mice in the NTR group and 2 out of 3 mice from the HSV-TK SR39 group died from progressive GvHD, while all of the mice in the iCasp9 group survived with no evidence of GvHD. In conclusion, our results suggest that the long-term safety and efficacy data of iCasp9, together with its lack of immunogenicity and the absence of reported side effects of CID (Di Stasi et al. ASH 2010 abstract #559), might be advantageous as a promising suicide gene-based strategy in ensuring the rapid and sustained eradication of the adoptively transferred T cells in case of unanticipated severe toxicities of cellular therapies. Based on this in vivo data, we are planning to incorporate the iCasp9 suicide gene in our future clinical trials with T cells genetically modified to express a chimeric antigen receptor targeted against a tumor specific antigen. Disclosures: No relevant conflicts of interest to declare.

Abstract 3767 Transplantation of unrelated umbilical cord blood (CB) derived stem cells is often used to treat adult patients with B-cell acute lymphoblastic leukemia (B-ALL). However, many patients relapse and overall prognosis is poor. We hypothesize that additional therapy involving adoptive transfer of CB derived T cells modified to express a CD19-specific chimeric antigen receptor (CAR) could improve patient outcome following allogeneic CB transplant. To this end, we have previously demonstrated that human T cells which express the anti-CD19 19–28ζ CAR, containing the signaling domains of the co-stimulatory CD28 receptor and CD3ζ chain, effectively eradicate CD19+ tumors both in vitro as well as in vivo in SCID-Beige mice. Herein, we demonstrate the ability to effectively isolate and expand T cells from CB samples using magnetic beads coated with agonistic CD3 and CD28 antibodies (Invitrogen) and comparing subsequent T cell expansions in in vitro cultures with varying additions of exogenous stimulatory cytokines, including IL-2, a combination of IL-2 and IL-7, IL-12 or IL-15. We demonstrate that in vitro culture in the context of exogenous IL-12 (10 ng/ml) resulted in optimal expansion of CB T cells (150-fold). In addition, expansion of T cells in the context of exogenous IL-12 resulted in a favorable phenotype for adoptive cell transfer, with T cells expressing high levels of Granzyme B and Perforin while retaining a “memory” phenotype as assessed by persistent expression of CD62L. This combination of cytotoxic and memory phenotype is optimal for adoptive cell therapy. T cells expanded in this manner were subsequently efficiently retrovirally transduced to express the 19–28ζ CAR. The resulting CD19-specific CB derived T cells were able to specifically lyse CD19+ tumor targets as assessed by standard 51Cr release cytotoxicity assays. We are currently investigating the in vivo anti-tumor function of these modified cells in our previously established preclinical SCID-Beige mouse tumor model and ultimately plan to conduct a Phase 1 clinical trial with these modified T cells in patients with B-ALL undergoing CB transplant. Disclosures: No relevant conflicts of interest to declare.

Abstract 3771 Adoptive infusion of T cells genetically modified to express chimeric antigen receptors (CARs) targeted to tumor associated antigens (TAAs) is a promising approach to cancer therapy. However, since TAAs are often expressed by normal tissues, safeguards are needed in the form of additional transduced suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events which may develop in the clinical setting. To this end, we have investigated the in vitro function of 3 different suicide genes each inserted distal to a CAR gene targeted against CD19 (19-28z) and a 2A linker peptide cloned into the SFG gammaretroviral vector. Specifically, we have tested the herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and the E.coli derived nitroreductase (NTR) with the prodrug metronidazole. Cell growth of PG13 murine fibroblasts transduced to express 19–28z CAR with NTR, HSV-TK, and iCasp9 was inhibited by 80% at 1mM of metronidazole, 85% at 1μM of ganciclovir, and 90% at 10nM of CID, respectively, when compared to control PG-13 fibroblasts. The drug concentrations tested in these assays were at physiologically achievable concentrations in humans, and did not affect the growth rate of control PG13 fibroblasts. Consistent with these findings in PG13 fibroblasts, we found that human T cells transduced with either 1928z.2A.NTR or 1928z.2A.HSV-TK demonstrated 90% and 88% inhibition, respectively, at similar substrate concentrations. Furthermore, we demonstrate that expression of these suicide genes does not affect the phenotype or function of the 19–28z CAR+ T cells, as assessed in vitro by T cell proliferation and cytotoxicity against CD19-expressing tumor cells. Our studies demonstrate highly effective suicide genes for human T lymphocytes transduced with a tumor targeted CAR, and a novel suicide gene/prodrug (NTR/metronidazole) combination with a comparable efficacy that can potentially serve as a reliable safety mechanism for adoptive T cell immunotherapy. While HSV-TK/ganciclovir has been utilized in various clinical settings, the NTR suicide gene has yet to be used in combination with gene modified tumor-targeted T cells. Furthermore, the NTR suicide gene holds several advantages over the HSV-TK and iCasp9 vectors. First, unlike HSV-TK, the NTR suicide gene is effective in both proliferating and non-proliferating cells. Second, unlike CID that is not commercially available, metronidazole is a widely available antibiotic that is relatively non-toxic. Lastly, metronidazole can be used in patients who may already be taking ganciclovir for cytomegalovirus (CMV) prophylaxis or treatment therefore limiting the application of T cells modified to express the HSV-TK suicide gene. Based on this in vitro data, we are currently testing the function of this suicide gene in vivo in two different animal models. Ultimately we anticipate that further studies with this novel suicide gene/prodrug combination will allow us to enhance safety in future clinical trials utilizing gene modified tumor targeted T cells. Disclosures: No relevant conflicts of interest to declare.