No abstract

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: 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.

T cells modified to express a chimeric-antigen receptor (CAR)targeting CD19 can induce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). The durability of remission is related to the length of time the CAR T cells persist. Efforts to understand differences in persistence have focused on the CAR construct, in particular the co-stimulatory signaling module of the chimeric receptor. We previously reported a robust intent-to-treat product manufacturing success rate and remission induction rate in children and young adults with recurrent/refractory B-ALL using the SCRI-CAR19v1 product, a 2nd generation CD19-specific CAR with 4-1BB costimulation co-expressed with the EGFRt cell surface tag (NCT02028455). Following completion of the phase 1 study, two changes to CAR T-cell manufacturing were introduced: switching the T-cell activation reagent and omitting mid-culture EGFRt immunomagnetic selection. We tested the modified manufacturing process and resulting product, designated SCRI-CAR19v2, in a cohort of 21 subjects on the phase 2 arm of the trial. Here, we describe the unanticipated enhancement in product performance resulting in prolonged persistence and B-cell aplasia, and improved leukemia-free survival with SCRI-CAR19v2 as compared to SCRI-CAR19v1.

Before and after bone marrow transplantation (BMT) for hematologic malignancies, peripheral blood mononuclear cells from 10 patients were obtained. The relative and absolute numbers of CD3+ T-cell receptor gamma delta+ (TCR gamma delta+) cells, as defined by the reaction of monoclonal antibodies (MoAbs) directed against CD3 and the TCR gamma delta (anti-TCR gamma delta-1), were determined. Before transplantation, eight of nine patients tested had less than 10% CD3+TCR gamma delta+ cells. Consistent increased numbers of gamma delta cells up to eightfold the pretransplant level can be seen in four of nine patients tested within the first 4 months after BMT. The large majority of early posttransplant gamma delta and alpha beta T cells express the CD45RO antigen, which is usually expressed on “memory” cells only. The V-region usage of the TCR gamma delta+ T cells was analyzed using fresh mononuclear cells and MoAbs against known V gamma and V delta regions. For more detailed analysis, CD3+TCR gamma delta+ cells were sorted and cultured in bulk and cloned. Using fresh cells and bulk cultures, mainly V gamma 9+V delta 1-V delta 2+ cells were found during engraftment. Only after 6 weeks post-BMT, V gamma 9-V delta 1+V delta 2- cells appear. Analysis of the V gamma and V delta usage at the clonal level confirmed the observation that early after BMT only V gamma 9+V delta 2+ cells are present, whereas gamma delta T- cell clones expressing other gamma delta TCR phenotypes can only be detected 4 to 6 weeks post-BMT. The predominance of V gamma 9+ cells during early engraftment could be explained by several mechanisms: (A) sequential rearrangements during T-cell development, leading to an early wave of V gamma 9+ cells, or (B) selective outgrowth of preexisting V gamma 9+V delta 2+CD45RO+ TCR gamma delta cells in the bone marrow graft, possibly as a result of antigen driven expansion due to exposure to environmental antigens.