T Cell Products of Defined CD4:CD8 Composition and Prescribed Levels of CD19CAR/Egfrt Transgene Expression Mediate Regression of Acute Lymphoblastic Leukemia in the Setting of Post-Allohsct Relapse
Authors: Gardner Rebecca A , Park Julie R , Kelly-Spratt Karen S , Finney Olivia , Smithers Hannah , Hoglund Virginia , Brown Christopher , Mgebroff Stephanie , Lindgren Catherine G. , Oron Assaf P , Bleakley Marie , Jensen Michael C 
Published: October 2019

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

Disclosures
Off Label Use: tociluzimab for treatment of cytokine release syndrome cetuximab for the ablation of T cells CAR T cells for the treatment of leukemia. Jensen:Juno: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.