Therapy of Paediatric B-ALL with a Fast Off Rate CD19 CAR Leads to Enhanced Expansion and Prolonged CAR T Cell Persistence in Patients with Low Bone Marrow Tumour Burden, and Is Associated with a Favourable Toxicity Profile

Abstract

Introduction: The CARPALL study (NCT02443831) employed a novel CD19CAR (CAT-41BBz CAR) with a faster off rate than the Kymriah FMC63-41BBz CAR (CAT 3.1x10-3s-1, FMC 6.8 x 10-5s-1), with equivalent on-rate (CAT 2.2 x 105, FMC 2.1 x 105). We herein report updated outcomes and CAR T cell persistence with an additional 6 months follow up from a submitted manuscript (Ghorashian et al., Nat Med, submitted) Methods: Patients aged <25 years with high risk, relapsed CD19+ B-ALL were eligible on this multi-centre, open label, non-randomised phase I study of autologous CAT-41BBz CAR T cells. Patients were followed to a data cut-off of 07/18/2019. CAT-41BBz CAR T cells were generated by magnetic bead activation of leucapheresed PBMCs, lentiviral transduction, followed by bioreactor expansion and magnetic bead removal prior to cryopreservation. All patients received lymphodepletion (fludarabine + cyclophosphamide) followed by 1x106/kg CAR T cells. Presence of CAR T cells in the blood and bone marrow (BM) was assessed (flow cytometry and qPCR) monthly for 6 months, then 6 weekly to 1 year and then 3 monthly. BM MRD was assessed (IgH qPCR, flow cytometry) at the same time-points up to 2 years to establish durability of responses as a stand-alone therapy. Primary end-points were incidence of grade 3-5 toxicity and the proportion of patients achieving molecular remission. Results: Of 17 patients recruited, 14 were treated due to manufacturing failure in 3 patients.The median age was 9 years (range 1-19 years). All patients had advanced ALL with a median of 4 prior therapy lines. 10 of 14 patients (71%) had relapsed post allogeneic SCT. Prior to lymphodepletion, 4 patients had >5% BM disease, 6 had disease between 5x10-2and 1x10-5, 4 were BM MRD negative having had recurrent isolated CNS disease. Median transduction efficiency was 31% (range 16.5 to 96.4%). 12/14 treated patients received the anticipated dose of 1x106CAR T cells/kg (2 received 0.9x106/kg). Considering all evaluable patients, (n=14 for CAR T cell persistence by qPCR, n=13 by flow) the geometric mean of Cmax was 128 912/µg DNA and of the area under the curve between D0 and D28 was 1,721,355 copies/ µg DNA (Table 1). At the point of maximal expansion, a median of 35% of circulating T cells were CAR+. Median half-life was 34 days (range 3-102). CAR T cells continued to be detectable by qPCR in 11 of 14 (79%) patients at last assessment and by flow cytometry up to 30 months post infusion in 8 of 13(61%). Median duration of CAR T persistence by flow was 261 days (range 7-917). 3 patients failed to have persistence of CAR T cells beyond 1 month. T cell mediated anti-CAR specific cytotoxic activity was detected in 2/2 evaluable patients. Updated persistence data will be presented at the meeting Cytokine release syndrome (CRS) occurred in 13 (93%, grade 1 n=9, grade 2 n=4). None developed ≥grade 3 CRS, had CRS-related ICU admission, or received Tocilizumab. CRS was associated with modest elevations of IL-6, IFN-γand IL-10. Grade 2 neurotoxicity was observed in 3 patients and resolved spontaneously. One patient had grade 4 leucoencephalopathy presumed due to chemotherapy as well as grade 5 sepsis. Ten patients (71%) had grade 3-4 cytopenia persisting beyond day 28 or recurring afterthis. 12/14 (86%) patients achieved molecular complete or continuing complete remission at a median of 30 days post infusion (range 30-90 days, Table 2). At a median follow-up of 20.3 months, 4/14 (29%) evaluable patients remain MRD negative. 5 relapsed with CD19-disease, 1 with CD19+ disease. The median duration of EFS (based on death or morphological relapse) has not been reached, 12 month EFS = 52%, OS = 70% (Figures 1, 2 and Table 3). Conclusion: We noted excellent CAR T cell expansion and persistence in a ALL cohort treated with the fast off-rate CAT-41BBz CAR despite their lower BM disease at treatment compared to other studies. The kinetics documented for all evaluable patients showed a 5-fold greater CAR T cell expansion and 2-fold longer half-life than responders in published series utilising tisagenlecleucel in a similar ALL cohort (Mueller et al., Blood 2017). Patients had a favourable toxicity profile with no severe (grade 3-4) CRS and equivalent disease outcomes to the ELIANA study despite having similarly advanced disease (Maude et al., NEJM 2018292). These data suggest long lived CAR T cell persistence supports stand-alone therapy for ALL with durable responses. Disclosures Ghorashian: Celgene: Honoraria; novartis: Honoraria; UCLB: Patents & Royalties: UCLB. Kramer:UCLB: Patents & Royalties. Ciocarlie:Servier: Other: Financial Support. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Amrolia:UCLB: Patents & Royalties.

High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells

“…The KD for a receptor is the ratio between how fast the receptor dissociates from its antigen, koff, and how fast the receptor binds to its antigen, kon. The koff for the FMC63 CD19 CAR is 6.8 x 10 -5 s -1 , whereas the koff for a TCR can vary from as fast as 10 -1 s -1 to as slow as 10 -3 s -1 , which is still over 100 times faster than that of the FMC63 CAR 40,72 . Seminal work showed that the longer a Treg is bound to a target dendritic cell, the more likely the Treg is to kill that cell 52 .…”

“…Our first aim was to investigate whether stimulation via a CAR or endogenous TCR/CD28 pathways results in different levels of Treg activation. Given the higher affinity of CARs, including the FMC63 scFv-based CD19 CAR used here 36,40 , compared to TCRs 41,42 , we hypothesized that CAR-mediated activation would lead to a heightened activation state. To assess this, CAR Tregs were coincubated with each K562 cell line, harvested after 48 hours, and their activation status was evaluated by measuring the cell surface expression of CD71 (transferrin receptor), a wellestablished early marker of T-cell activation.…”

“…Inspired by these notions, we modified our CAR construct to dissect which of its features was responsible for the proinflammatory shift observed in CAR-activated Tregs and potentially better mimic endogenous TCR/CD28 engagement in Tregs. To reduce affinity, we modified the extracellular domain of the CAR by swapping the FMC63 scFv domain with an scFv sequence, CAT-13.1E10, which binds to the same CD19 residues as FMC63 but with a 40-fold lower affinity 40 . To reduce costimulation strength, we modified the intracellular domain of the CAR by mutating all tyrosines of the CD28 signaling domain, as well as both prolines of its PYAP domain, which binds to Lck 31,74 .…”

Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells

“…The authors reasoned that an scFv targeting ALK with favorable binding kinetics for immune synapse stabilization might be one solution to allow signal strength to achieve a threshold for full effector function of ALK CAR T cells against antigen-low neuroblastoma. Recent studies into the relationship of antibody affinity with CAR T-cell function have led to some generalizable findings, such as the observation that fast offrate can lead to short dwell time and potentially diminish T-cell exhaustion [26,27]. However, the precise rules governing the relationship between binding kinetics and quality and quantity of T-cell signal have yet to be elucidated, and the authors therefore followed an empirical approach of screening a panel of CAR T-cell constructs by effector function.…”

Dynamic regulation of hierarchical heterogeneity in Acute Myeloid Leukemia serves as a tumor immunoevasion mechanism

“…Despite efforts to manipulate donor HSCs to improve transplantation outcomes (Fares et al, 2014;Goncalves et al, 2016;Grey et al, 2020;Holmfeldt et al, 2016), accumulating data show that post-transplant relapse correlates with T cell exhaustion or immune-edited variants lacking major histocompatibility antigen expression (Christopher et al, 2018;Noviello et al, 2019;Toffalori et al, 2019). Immunotherapies have been very promising for malignancies such as melanoma (Leonardi et al, 2020;Luke et al, 2017) and B-cell Acute Lymphoblastic Leukemia (Ghorashian et al, 2019;Maude et al, 2018;Park et al, 2018), but have so far produced disappointing results against AML. Although anti-tumor immune responses have been reported in a variety of AML murine models and in AML patients (Hayashi et al, 2019;Rezvani et al, 2005;Zhang et al, 2009;Zhou et al, 2011), paucity of AML-specific antigens, limited in vivo persistence of adoptively transferred T cells, and inability to precisely identify patients likely to benefit have limited immunotherapeutic approaches (Witkowski et al, 2019).…”