Gardner et al report that early intervention with tocilizumab and steroids at the first signs of mild cytokine release syndrome (CRS) following CD19 chimeric antigen receptor (CAR) T-cell infusion for B-cell acute lymphocytic leukemia reduces the development of life-threatening severe CRS without having a negative impact on antileukemic effect.

Mononuclear cell apheresis for chimeric antigen receptor T-cell therapy is well tolerated and safe, and it is possible to obtain an adequate quantity of CD3+ lymphocytes for chimeric antigen receptor T-cell manufacturing in heavily pretreated patients who have low lymphocyte counts.

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

Introduction: Advances in chimeric antigen receptor (CAR) T cell therapy have yielded complete remission (CR) rates in relapsed/refractory B-ALL (rrB-ALL) of 70-95%. However, disease recurrence after CD19 or CD22 CAR therapy is greater than 50% at 1 year, and approximately half of recurrences are due to antigen escape. To reduce antigen escape and optimize the durability of remission, we sought to design a CAR T cell product with dual specificity that is capable of simultaneously targeting both CD19 and CD22. Preclinical testing of our bi-specific CAR showed a preference for signaling through CD22 over the CD19 CAR. In contrast, dual transduced T cells signaled through both the CD19 and CD22 CAR with lytic activity and cytokine production similar to single transduced CAR T cells of the same specificity. Therefore, we opted to move forward with dual transduced T cells for clinical use. We are currently testing SCRI-CAR19x22v1 in PLAT-05 (NCT03330691), a phase 1 clinical trial for pediatric and young adult patients with CD19+CD22+ rrB-ALL, with the primary objectives to determine the feasibility of manufacturing products with dual specificity, to assess the safety of the cryopreserved product infusion, and to describe the full toxicity profile. Methods: Subjects undergo apheresis, after which the CD4 and CD8 T cell subsets are immunomagnetically selected and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two separate SIN lentiviral vectors that direct the expression of a CD19-specific FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR with an Her2tG tag and expression of a CD22-specific m971scFv:IgG4hinge:CH2(L235D)-CH3:CD28tm:4-1BB:ζ CAR with an EGFRt tag, creating three distinct populations of CAR T cells (anti-CD19, anti-CD22, and anti-CD19x 22). Transduced cells are expanded in serum free media formulation with IL-7, IL-15, and IL-21. Following lymphodepleting chemotherapy, cryopreserved products are thawed and infused at the protocol-prescribed dose level. Cytokine release syndrome (CRS) is graded according to Lee et al. (Blood 2014) and is treated according to our early intervention strategy of tocilizumab and dexamethasone for persistent, mild CRS. Results: Seven subjects (ages 1-26 yr) with rrB-ALL have been enrolled with 4 treated at dose level 1 (1 x 106 CAR T cells/kg) and 3 treated at dose level 2 (3 x 106 CAR T cells/kg). The mean culture time was 7.9 days (range 7-11) and subjects received infusions with a mean CD8:CD4 ratio of 1.7 (range 0.2 - 3.1). CD8 CAR composition, on average, consisted of 21.6 % CD19 CAR, 37.8 % CD22 CAR, and 40.6 % CD22xCD19 CAR T cells. CD4 CAR composition, on average, consisted of 25.8 % CD19 CAR, 30.6 % CD22 CAR, and 43.6 % CD22xCD19 CAR T cells (Figure). Peak engraftment occurred between days 7 and 14 for all patients and was predominantly composed of the CD19 CAR population with median peak values for CD19 CAR, CD22 CAR, and CD19xCD22 CAR T cell populations of 9.1%, 1.2%, and 2.4%, respectively. A CR was achieved in 5/7 (71%) subjects by day 21, 4 of which were minimal residual disease negative. The two subjects without a CR did not exhibit evidence of CAR T cell engraftment; one had previously received CD19 CAR T cells, and the other had progressive disease and pursued alternative therapy at day 10. Therapy was well tolerated with no dose limiting toxicities. CRS occurred in 5 subjects (Grade 1) with 2 of these subjects experiencing mild neurotoxicity (Grade 1). Four subjects received tocilizumab +/- dexamethasone, and two of these received multiple doses of dexamethasone. Conclusions: Preclinical testing showed superior efficacy against both CD19 and CD22 when using two separate CARs and dual transduction, compared to a single bi-specific CAR. Preliminary analysis of PLAT-05 supports feasibility of product manufacturing, and toxicity and response rates that are consistent with the reported CD19 CAR T cell experience. While the infused SCRI-CAR19x22v1 products consist of a near-uniform distribution of the 3 distinct populations, we observed selective in vivo expansion of the CD19 CAR T cell population. Further investigation is required to understand the mechanism of CD19 CAR dominance in vivo. Continued accrual of subjects is ongoing to further assess the impact of dual antigen targeting on the prevention of antigen escape and the potential to provide a more durable remission. Figure. Figure. Disclosures Park: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Background: The youngest patients referred for CAR T cell therapy are those with relapsed or refractory (R/R) KMT2A-rearranged infant B-ALL. Infants with relapsed ALL following Interfant-99 therapy have a dismal reported 3-yr OS of 20.9%, indicating the need for novel therapies. Smaller patient size, heavily pre-treated disease and high leukemia burden are often characteristics of this subgroup of patients that pose unique challenges to apheresis and manufacture of a T cell product. Additionally, reports of KMT2A-rearranged leukemia undergoing lineage switch following CD19-targeting pressure raises concern for an increased risk of myeloid leukemia relapses after B-lineage targeted CAR T cell therapy in this population. Here we report our experience using CAR T cell immunotherapy for patients with R/R infant ALL enrolled on clinical trials PLAT-02 (NCT02028455) and PLAT-05 (NCT03330691). Methods: PLAT-02 is a phase 1/2 trial of CD19-specific (FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ) CAR T cells. PLAT-05 is a phase 1 trial of CD19xCD22 dual specific CAR T cells, transduced with two separate lentiviral vectors to direct the co-expression of the CD19-specific CAR above and a CD22-specific CAR (m971scFv:IgG4hinge-CH2(L235D)-CH3-CD28tm:4-1BB:ζ). Eligible subjects on both studies have R/R B-ALL, an absolute lymphocyte count ≥100 cells/µL, and were at least 1 year of age. In addition, subjects on PLAT-02 were ≥ 10kg, and ≥ 8kg on PLAT-05. For cell manufacture, apheresis products were immuno-magnetically selected for CD4 and CD8 cells. Selected T cells were activated with anti-CD3/CD28 beads, transduced, and grown in culture with homeostatic cytokines to numbers suitable for clinical use. Infant ALL subjects received a range of 5x105 to 10x106 CAR+ T cells/kg following lymphodepleting chemotherapy. Disease response assessments were required at Day 21 and Day 63 following CAR T cell infusion. Adverse events were graded according to CTCAEv4, except CRS which was graded according to 2014 Lee criteria. Results: Eighteen subjects with R/R infant ALL have enrolled on PLAT-02 (n=14) or PLAT-05 (n=4), with a median age of 22.5 months at enrollment (range: 14.5 - 40.1 months). Of these, 2 (11.1%) had primary refractory disease, 8 (44.4%) were in 1st relapse, 7 (38.9%) were in 2nd relapse and 1 (5.6%) was in 3rd or greater relapse. Ten subjects (55.6%) had an M2 marrow or greater at enrollment prior to apheresis, and 9/18 had a history of hematopoietic cell transplant (HCT). The mean ALC was 1309 cells/µL (range 253-6944). Successful CAR T cell products were manufactured in 17/18 subjects, including in 9/9 subjects with no prior history of HCT. Of these, 16/17 subjects with available products were infused, with a median follow up of 26.9 months. One subject died of disease complications prior to CAR T cell infusion. Of the 16 treated subjects, 1 is pending disease and toxicity assessments. The maximum grade of CRS was 3 and occurred in two of 15 evaluable subjects (13%) and neurotoxicity was limited to a maximum grade of 2. Fourteen of 15 (93.3%) achieved an MRD negative complete remission (MRD-CR) by Day 21. Of the 14 subjects with an MRD-CR, 6 went on to HCT with 1 subsequent CD19 negative relapse. Of the 8 subjects who did not proceed to HCT, 1 developed lineage switch at one month following CAR T cells, and 1 died of infectious complications with aplasia. A "wait and watch" approach was taken for the remaining 6 subjects, and 2 developed CD19+ relapse. The incidence of lineage switch among the infant ALL group was 1/15 (6.7%). The estimated 1-year LFS was 66.7% and 1-year OS was 71.4%. Conclusion: This is the largest reported cohort to date of R/R infant B-ALL subjects treated with CAR T cell therapy. We report successful manufacture and administration of a CAR T cell product in the significant majority of infant subjects. Toxicity and MRD-CR rates are comparable to that of non-infant ALL subjects. In our experience, subjects with R/R infant ALL are not at increased risk for lineage switch relapse compared with the entire study populations following B-antigen targeting CAR T cell immunotherapy. Numbers in this report are too small to make definitive conclusions about the value of consolidative HCT. However, the LFS of this cohort is remarkably higher when compared with historical controls. Future work is focused on overcoming feasibility issues for the smallest of subjects, to enable a larger number of these cases to access CAR T cell therapy. Disclosures Pulsipher: Amgen: Other: Lecture; Bellicum: Consultancy; Miltenyi: Research Funding; Medac: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Wayne:AbbVie: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding; Servier: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding. Jensen:Bluebird Bio: Research Funding; Juno Therapeutics, a Celgene Company: Research Funding. Gardner:Novartis: Honoraria.

Background:Pediatric patients with relapsed or refractory CD19+non-Hodgkin lymphoma (NHL) have poor outcomes despite use of chemotherapy and hematopoietic stem cell transplant (HSCT). Clinical trials of CD19 CAR T-cells have demonstrated efficacy in salvaging adult patients with relapsed and refractory NHL. Objectives:The objectives of this analysis is to assess the safety, toxicity, feasibility and efficacy of SCRI-CAR19v1 for pediatric patients with relapsed or refractory NHL. Design/Methods:The ongoing phase 2 trial (NCT02028455) has enrolled and treated 8 pediatric subjects with CD19+NHL. Subjects underwent apheresis, with their CD4 and CD8 T cell subsets prepared immunomagnetically. T cells were stimulated with anti-CD3xCD28 bead stimulation, and then transduced with a SIN lentiviral vector to direct co-expression of the FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR and the selection/tracking/suicide construct EGFRt. The transduced cells were propagated using recombinant human cytokines to numbers suitable for clinical use. Subjects received lymphodepletion of fludarabine and cyclophosphamide followed by 1x106CD19 CAR T-cells/kg as a 1:1 ratio of CD4 and CD8 cells. Response was assessed at 3 and 9 weeks. Adverse events were graded according to CTCAEv4 except cytokine release syndrome (CRS) was graded according to Lee et al. Results: Treated subjects had relapsed or refractory diffuse large B cell lymphoma (DLBCL) (4/8), Burkitt's lymphoma (2/8), gray zone B cell lymphoma (1/8), primary mediastinal B cell lymphoma (PMBCL) (1/8), and ranged from 4-18 years old. Two subjects received prior hematopoietic stem cell transplant (HSCT); the subject with PMBCL received auto- and allogeneic HSCT and a subject with Burkitt's received autologous HSCT. Five subjects received prior immunotherapy with brentuximab, nivolumab, rituximab, and/or obinutuzumab. One subject had received ibrutinib. No subject had received prior CAR T-cells. CD4 and CD8 products were successfully manufactured and infused for all subjects. All subjects had expansion of CAR T-cells in the peripheral blood, bone marrow and CSF, with ongoing persistence at last check (range 14 days - 9 months). Toxicity information through day 30 revealed the occurrence of mild CRS in 4 subjects (grade 1 n=3, grade 2 n=1), and one case of severe CRS (grade 3). Mild neurotoxicity was observed in 2 subjects (grade 1 n=1, grade 2 n=1) with no occurrence of severe neurotoxicity. Response assessment at 3 weeks (n=6) revealed anti-tumor responses in 5 subjects, including complete response (CR) by week 9 (n=2, both DLBCL). CR was not sustained in either subject despite ongoing CAR T cell persistence. One of these subjects had a PET avid lesion proven by biopsy to be necrotic tissue but subsequently developed CD19+recurrence at that site. The other subject developed a new CD19+site of disease at six months; however, achieved a 2nd CR 3 weeks after receiving a second infusion of the originally manufactured CAR T-cells. One partial response (PR) subject experienced clearance of marrow disease with stable lymphoma but developed CD19 negative progression at 9 weeks. Updated enrollment, toxicity and response assessments will be presented. Conclusion:SCRI-CAR19v1 therapy demonstrates efficacy in pediatric patients with relapsed and refractory NHL and appears to be well tolerated with less severe toxicities than observed for pediatric patients with CD19+leukemia. Persistence of the CAR T-cells is excellent with no early loss of CAR T-cell engraftment reported to date. Although early responses were observed, these were not durable perhaps reflecting biologic/immunologic differences between B cell lymphomas in children in comparison to NHL in adults. Disclosures Pulsipher: Adaptive Biotech: Consultancy, Research Funding; Amgen: Honoraria; CSL Behring: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Park:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Background: While immunotherapy withCD19 specific CAR T cells for relapsed/refractory (R/R) B-ALL achieves MRD negative remission in nearly all patients, relapse occurs in approximately half of patients and is frequently associated with early loss of CAR T cell persistence. Low CD19 antigen burden in the bone marrow prior to lymphodepletion and rapid contraction of CAR T cells in the blood after engraftment are predictive of early loss of CAR T cell persistence. We hypothesize that episodic antigen exposure using CD19t T cell antigen presenting cells (T-APCs) can trigger CD19 CAR T cell proliferation and re-activation in vivo, resulting in more durable CAR T cell persistence and diminished risk of CD19+ relapse. Here we report our experience to date using T-APCs following CD19 CAR T cell therapy for children and young adults with R/R B-ALL on clinical trial PLAT-03 (NCT03186118). Methods: Patient-derived T-APC products were manufactured from cryopreserved CD4/CD8 selected T cells. Cells were then activated with anti-CD3/CD28 beads, transduced with a lentiviral vector to express a truncated human CD19 (CD19t), and expanded in culture for 10 days. Subjects <25kg received 10x106 T-APCs/kg/dose and those ≥25kg received a flat dose of 5x108 T-APCs/dose, for up to a total of six-monthly doses of T-APCs. Adverse events were graded according to CTCAEv4. Results: Fourteen subjects (8-26 yrs) have been enrolled; 8 with low CD19 antigen burden, 5 with rapid CAR T cell contraction, and 1 subject with early loss of CAR T cell persistence on a predecessor study who received a re-infusion of CD19 CAR T cells on this study followed by T-APCs. T-APCs were successfully manufactured in 14/14 subjects. Two subjects lost CAR T cell persistence prior to T-APC infusion and were ineligible to receive T-APCs. To date, 11 subjects have received at least one dose of T-APCs. One of 11 subjects experienced a grade 3 febrile infusion reaction within hours of the 2nd dose of T-APCs, prohibiting further dosing. There were no other related adverse events (AEs) > grade 2 among the 11 subjects, and no cytokine release syndrome or neurotoxicity has been observed. An increase in detectable CD19 CAR T cells occurred in all subjects following T-APCs, and T-APCs can be transiently detected following infusion (Figure 1). Of the 10 treated subjects with low CD19 antigen burden or rapid T cell contraction, 8/10 had CAR T cell persistence beyond Day 63, as evidenced by B cell aplasia. At last follow-up, 5/10 have ongoing B cell aplasia, with a median follow up of 8.8 months (range, 2-18.5 months). The estimated 1-year leukemia free survival (LFS) is 69.2%. Conclusion: This first-in-human study of CD19t T-APCs demonstrates the ability to successfully manufacture T-APCs from stored apheresis products collected for CAR T cell production. In 11 subjects receiving at least one T-APC dose to date, there has been one T-APC infusion reaction and no other significant associated toxicity. Early evidence of efficacy demonstrated by secondary expansion of CAR T cells suggests the potential of CD19t T-APCs to enhance durable CD19 CAR T cell persistence. Figure 1 Disclosures Gardner: Novartis: Honoraria. Jensen:Bluebird Bio: Research Funding; Juno Therapeutics, a Celgene Company: Research Funding.

Burkitt lymphoma arising in paediatric post-solid-organ transplantation-Burkitt lymphoma (PSOT-BL) is a clinically aggressive malignancy and a rare form of posttransplant lymphoproliferative disorder (PTLD). We evaluated 35 patients diagnosed with PSOT-BL at 14 paediatric medical centres in the United States. Median age at organ transplantation was 2.0 years (range: 0.1-14) and age at PSOT-BL diagnosis was 8.0 years (range: 1-17). All but one patient had late onset of PSOT-BL (≥2 years post-transplant), with a median interval from transplant to PSOT-BL diagnosis of 4.0 years (range: 0.4-12). Heart (n = 18 [51.4%]) and liver (n = 13 [37.1%]) were the most frequently transplanted organs. No patients had loss of graft or treatmentrelated mortality. A variety of treatment regimens were used, led by intensive Burkitt lymphoma-specific French-American-British/Lymphomes Malins B (FAB/ LMB), n = 13 (37.1%), and a low-intensity regimen consisting of cyclophosphamide,