Frey, Noelle V.

Background: Chimeric antigen receptor (CAR) T cells have repeatedly demonstrated capacity to induce high rates of response and durable remissions for B cell cancers. In contrast, CAR T cells for the treatment of acute myeloid leukemia (AML) have not yet achieved this impact. We previously demonstrated that CD123 is expressed on AML blasts at high frequency, thus making it an attractive target antigen for AML-directed CAR T cells. Methods and Results: We conducted a pilot study of CD123-directed CAR T cells (CART-123) in adults with relapsed or refractory AML. The primary objective was safety with a secondary objective of anti-leukemia efficacy. Twenty-two subjects were screened, and 20 were eligible for the trial. Fludarabine and cyclophosphamide were used for lymphodepletion (LD). Twelve subjects were infused with CART-123. There were two treatment-related deaths due to cytokine release syndrome (CRS) and infection. Four subjects (33%) achieved complete response with incomplete count recovery (CRi), three subjects had progressive disease, and five had stable disease. Of the four who achieved CRi, one subject remains alive in remission after a relapse and subsequent consolidative hematopoietic stem cell transplant (HSCT). One other subject is alive after HSCT following CART-123 failure. There was evidence of CAR T cell expansion and ten subjects (83%) experienced clinical CRS ranging from Grade I to Grade V. There was no neurotoxicity. To address the apparent paradox of high rates of CART-123 bioactivity (clinical CRS and in vivo expansion) co-occurring with low rates of response, we hypothesized that cytokines produced during CRS may undermine the anti-AML effect of CART cells. We obtained serum drawn from trial subjects at baseline (prior to LD chemotherapy) or during CRS. Serum drawn during CRS promotes primary AML survival in cell culture, while matched baseline serum from the same subjects does not. Individual cytokines secreted during CRS that signal through receptors expressed on myeloid progenitors (such as GM-CSF and Flt3 ligand) reproduced this survival advantage in primary AML blasts when tested at physiologically relevant levels. In contrast, these cytokines did not produce a pro-survival effect in primary patient B-ALL blasts. Crucially, candidate myeloid-acting cytokines, as well as serum drawn during the peak of clinical CRS, promoted resistance of AML to killing by CART-123 in vitro. Using scRNAseq of bone marrow aspirates from subjects on this clinical trial, we found that GM-CSF and FLT3L were most frequently expressed by CD4+ T cells. Exposure of primary AML samples to CRS cytokines led to upregulation of signaling (e.g. JAK/STAT5) and pro-survival pathways. In previous work in B-ALL, we showed that relative resistance to killing can translate to a functional deficit in CART-19 due to development of exhaustion in the CART cells. We similarly find that that exposure to GM-CSF reduces AML blast sensitivity to CART-123, and that the persistence of antigen leads to exhaustion in CART cells (Figure 1). Specifically, co-incubation of CART-123 cells and cytokine-exposed primary AML blasts leads to increased populations of PD-1+/CD39+/CTLA4+/LAG3+ CAR T cells, consistent with an exhaustion phenotype. Finally, we found that anti-apoptotic effects of cytokines can be prevented, and CART-123 killing of AML can be restored, via ruxolitinib blockade of JAK/STAT signaling in both in vitro and in vivo settings. Significance: Our work reveals CRS as active in undermining CART-123 lethality first by promoting survival of AML, and subsequently by exhausting CAR T cells. This is in stark contrast to its role in lymphoid malignancies and underscores biological differences in myeloid versus lymphoid neoplasms. These results highlight a need to tailor therapy to limit cytokine effects on AML blasts to improve outcomes with AML-directed CAR T cell therapy.

Introduction: ECOG ACRIN E1910 is a randomized phase III trial that showed that adults with newly diagnosed BCR::ABL1 negative acute lymphoblastic leukemia (ALL) who become MRD negative (<0.01%) after induction chemo who receive blinatumomab with conventional chemotherapy (chemo) have improved survival compared with those who received chemo only (Litzow et al, Blood (2022) 140: Supplement 2, LBA-1). However, not all pts were able to receive all four planned cycles of blinatumomab in consolidation. In this report we assessed outcomes of pts in the blinatumomab arm of the trial who received all 4 cycles of blinatumomab compared to those who received 1-2 cycles. Methods: Patient between the ages of 30 and 70 with newly diagnosed BCR::ABL1 negative B-lineage ALL were enrolled and initially received 2.5 months of combination induction chemo utilizing a BFM-like regimen adapted from the E2993/UKALLXII clinical trial, with pegaspargase (after induction only for patients >=55 years of age) and addition of rituximab for CD20 positive patients. After remission induction (step 1) pts in morphologic complete remission (CR/CRi) received an intensification course of high dose methotrexate with pegaspargase for CNS prophylaxis (step 2). At the conclusion of step 2, remission and MRD status were determined centrally by 6-color flow cytometry with MRD negativity defined as <0.01%. In the primary analysis subset, MRD negative patients were randomized to receive an additional 4cycles of consolidation chemo or 2 cycles of blinatumomab at 28 mcg/day (flat dose) for 28 days each cycle followed by 3 cycles of consolidation chemo, another 4-week cycle of blinatumomab (3rd cycle of blinatumomab) followed by an additional cycle of chemo and then a 4th cycle of blinatumomab (step 3). Following completion of consolidation chemo +/- blinatumomab, pts were given 2.5 years of POMP maintenance therapy timed from the start of the intensification cycle (step 4). Estimates of OS were calculated using the Kaplan-Meier method. Comparison of OS between treatment arms were conducted using the stratified log-rank test and Cox proportional-hazards model with age, CD20 status, rituximab use, and whether pts intend to receive HSCT or not as stratification factors. As the number of cycles needed to optimize outcome has not been studied, an unplanned landmark analysis was used to compare OS of pts in the blinatumomab arm who received all 4 cycles o those who received 1-2 cycles. Time 0 was chosen as 9 months post step 3 randomization (the time that patients were supposed to complete 4 cycles of blina). Four pts who received blinatumomab but died within 9 months post step 3 randomization were therefore excluded from this analysis. Results: The study was activated on December 17, 2013 and closed to enrollment on October 15, 2019. 772 pts were screened for the trial and 488 were enrolled on step 1 induction therapy. The median age of the pts was 51 years (range 30-70). 224 MRD negative pts were randomized, 112 pts to each arm. In the blinatumomab arm 12 pts received 1 cycle (11%), 32 pts received 2 cycles (29%), 4 pts received 3 cycles (4%) and 63 pts received 4 cycles (57%). The OS of pts who received 1-2 cycles of blinatumomab was compared to control (Figure 1) The difference was not significant (hazard ratio 0.62, 95% CI 0.28 to 1.34, p=0.22). Fig 2 compares the survival of those who received 1-2 cycles to those who received 4 cycles ( (HR: 0.39, 95% CI 0.12 to 1.16, p=0.076). Conclusion: The addition of blinatumomab to consolidation chemotherapy resulted in a significantly better overall survival compared to chemotherapy alone in pts with newly diagnosed B-lineage ALL who were MRD negative after intensification. This represents a new standard of care for pts aged 30-70 years with BCR::ABL1 negative ALL. The optimal dose and number of cycles is however unknown. In this unplanned subgroup analysis we demonstrate that a survival benefit can only be confirmed in patients who receive the intended 4 cycles of blinatumomab during consolidation. Further studies will be needed to determine if fewer cycles can provide a similar benefit.

Introduction: Cryopreservation of peripheral blood stem cell (PBSC) or bone marrow (BM) grafts is rarely performed, thus information about the effect of cryopreservation on graft characteristics and HCT outcomes is limited. Given the global pandemic leading to changing practices for donor graft collection and increasing utilization of cryopreservation, we evaluated the outcomes of HCT recipients who received either fresh or cryopreserved allogeneic BM or PBSC grafts. The primary endpoint was engraftment. Secondary endpoints were incidence of acute graft-vs.-host disease (aGVHD), relapse, transplant related mortality (TRM), disease free survival (DFS), and overall survival (OS). Methods: We included 7397 patients transplanted between 2013 and 2018. 1883 cryopreserved graft recipients were divided into three cohorts based on graft source and donor type: matched related (MRD) PBSC (n=1,051), matched unrelated (MUD) PBSC (n=678), and matched related or unrelated bone marrow donors (n=154). All patients received conventional calcineurin-based GVHD prophylaxis strategies. Patients who received cryopreserved grafts were matched with 5514 patients who received fresh adjusting for graft type (BM vs. PB), donor source (MRD vs. 8/8 MUD), Disease Risk Index (DRI, low risk vs. intermediate risk vs. high risk), recipient age (within 5-years) and propensity score (within 1 standard deviation from pooled sample). The propensity score was based on age, Karnofsky score, diagnosis, disease risk index, HCT-comorbidity index, and conditioning intensity. The level of statistical significance for the main analyses was p<0.01 due to multiple comparisons. The reason for cryopreservation was available on a subset of URD product recipients. Results: Baseline characteristics for all cohorts are shown in Table 1. CD34+ doses reflect enumeration at infusion. In univariate analyses, rates of graft failure and neutrophil recovery at day 28 were similar for cryopreserved and fresh grafts for BM or related PBSC recipients but differed in cryopreserved vs. fresh MUD PBSC (5% vs 2%, p<0.001 and 87 vs 94%, p<0.001, respectively). Rates of platelet recovery within 28 days were similar in marrow cohorts, but lower with cryopreserved related (92% vs 96%, p<0.001) and MUD PBSC (87 vs 94%, p<0.001) grafts. In matched pair multivariable analyses, there were no significant differences in any outcomes with cryopreserved vs fresh BM grafts, irrespective of donor type. With related donor PBSC, cryopreservation was associated with decreased platelet recovery (HR=0.73, CI=0.68-0.78, p<0.001) and an increased risk of both grade II-IV (HR=1.27, CI=1.09-1.48, p=0.002) and grade III-IV (HR=1.48, CI=1.19-1.84, p<0.001) aGVHD, but had no impact on other outcomes. In contrast, with MUD PBSC grafts, cryopreservation was associated with delayed engraftment of neutrophils (HR=0.77, CI=0.71-0.84, p<0.001) and platelets (HR=0.61, CI=0.56-0.66, p<0.001), an increased risk of TRM (HR=1.4, CI=1.18-1.66, p<0.001) and relapse (HR=1.32, CI=1.11-1.58, p=0.002), and decreased DFS (HR=1.36, CI=1.20-1.55, p<0.001) and OS (HR=1.38, CI=1.22-1.58, p<0.001) but did not affect the incidence of aGVHD. In a subset analysis of 299 URD recipients, the most common reason for cryopreservation was delays due to patient-related events, eg., additional chemotherapy, infection. These recipients had decreased OS compared to products cryopreserved for non-patient reasons (HR=0.65, CI=0.44-0.96, p=0.029). Conclusions: The retrospective nature of this analysis, and the fact that cryopreservation is likely to have been performed in recipients with a different (higher) risk profile compared to those receiving fresh products limits our ability to draw firm confusions. Despite these limitations, we conclude that cryopreservation in some patient populations may have a negative impact on engraftment and transplant outcomes. The decision to cryopreserve donor grafts, particularly PBSC grafts, should be carefully considered and highlights the need for further investigation of the effect of cryopreservation of allogeneic grafts where patient factors can be controlled for. Disclosures Farhadfar: CSL Behring: Research Funding; Incyte pharmaceutical: Other: Member of GVHD advisory forum. Frey:Kite Pharma: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Syntax: Consultancy, Honoraria. Devine:Magenta Therapeutics: Consultancy. Shaw:Orca Bio: Consultancy. Wingard:Shire: Other: Personal Fees - serve on data monitoring committee for clinical trial; Ansun: Other: Personal Fees - serve on data monitoring committee for clinical trial; Merck: Other: Personal Fees - serve on data monitoring committee for clinical trial; Cidara: Other: Personal Fees - serve on data monitoring committee for clinical trial; ReViral: Other: Personal Fees - serve on data monitoring committee for clinical trial.

Abstract 5-azacitidine (5-AZA) is standard of care in the first-line setting for higher-risk patients with Myelodysplastic Syndrome (MDS). However, 40-50% of patients are refractory to 5-AZA and most responders ultimately demonstrate disease progression within 2 years, often progressing to AML. For patients refractory to 5-AZA, 2-year survival probability is low (15%). Thus, there is a need for more effective initial and second-line therapies for patients with higher-risk MDS. The Inhibitors of Apoptosis (IAP) Proteins are a family of molecules that suppress apoptotic cell death. Expression of IAPs is dysregulated in MDS, suggesting a potential path for therapeutic intervention. SMAC (second mitochondrial activator of caspases) is an IAP antagonist, resulting in caspase activation, inhibition of NF-kB and increased apoptosis. Birinapant is a potent bivalent SMAC mimetic with improved tolerability and therapeutic index compared to other SMAC mimetics. This Phase Ib/2a study was designed primarily to determine the MTD and recommended Phase 2 dose and schedule of birinapant in combination with 5-AZA in patients with higher-risk MDS who are naïve or have relapsed or are refractory to 5-AZA. To date, 11 patients have been enrolled in the Phase 1b portion of the study, including 7 with IPSS classification high-risk MDS (5 male, 2 female; median age 75, range 64-82) and 4 with intermediate-risk MDS (2 male, 2 female; median age 72.5, range 65-84). All patients received standard 5-AZA, administered via either intravenous (IV) or subcutaneous (SC) routes, at 75 mg/m2 for 7 days of a 28-day cycle in combination with birinapant. Birinapant was administered IV twice weekly (Days 1 and 4) for either 3 of 4 weeks or 4 of 4 weeks per 28-day cycle at a dose of 13 mg/m2. No cycle 1 dose-limiting toxicities have been observed. Adverse events ≥ Grade 2 observed in two or more patients and judged to be related to treatment were rash/pruritis (Grade 2, 2 patients), thrombocytopenia (Grade 4, 2 patients) and neutropenia (Grade 4, 1 patient; Grade 3, 1 patient). Two SAEs, Grade 3 abdominal soft tissue necrosis with abdominal pain in the area of subcutaneous injections of 5-AZA, and Grade 3 cellulitis were judged to be related to treatment. These local injection site reactions/cellulitis were more severe than usually expected with SC 5-AZA, suggesting an on target pharmacodynamic (TNF mediated) localized synergistic effect in skin, but were not observed with IV administration. Both occurred after cycle 1 and were not considered DLTs. Data showing inhibition of NF-kB, a downstream pro-survival molecule activated by IAPs, in circulating blast cells from patients treated with birinapant at a dose of 13 mg/m2 twice weekly for 3 weeks out of 4, suggests that birinapant is pharmacologically active at this dosing schedule. Based on these and other pharmacodynamic data, despite not determining the formal MTD, the Phase 2 dosing regimen has been established as birinapant administered at 13 mg/m2 IV twice weekly for 3 weeks of a 28-day cycle in combination with 5-AZA at 75 mg/m2 by IV infusion for 7 days of a 28-day cycle. Although not a primary endpoint, evidence of clinical activity in 5-AZA resistant/refractory patients was observed in 2 of 11 patients to date, with an additional good clinical response in a 5-AZA naive high-risk MDS/CMML patient. One patient with prior 5-AZA-refractory MDS (cytogenetics: hypodiploid and +11q) showed bone marrow (BM) blast count reduction from 25% to 2% after 1 cycle of 5-AZA/birinapant treatment. Another patient with MDS (cytogenetics: +X; +19; +21) refractory to single-agent 5-AZA showed BM blast count reduction from 21% to 7% after 2 cycles. A third patient with treatment-naïve MDS/CMML-2 (cytogenetics: 46, XY, inv(6)(p11.2q15), t(6;21)(q21;q22) ) who had received prior hydroxyurea demonstrated BM blast count reduction from 17% to 2% after 3 cycles and underwent hematopoietic stem cell transplant. Several patients remain on study and are in active follow-up. This Phase 1b trial provide data for an acceptable safety profile of the first combination of a SMAC mimetic (birinapant) administered with IV 5-AZA. Early signs of clinical activity including in 5-AZA naïve and refractory patients were observed. These results provide rationale for a randomized Phase 2 study comparing birinapant in combination with 5-AZA against 5-AZA alone in the first-line setting for patients with higher-risk MDS. Disclosures Borthakur: Tetralogic Pharmaceuticals: Research Funding. Foran:TetraLogic Pharmaceuticals: Research Funding. Wang:Tetralogic Pharmaceuticals: Research Funding. Rakkar:Tetralogic Pharmaceuticals: Research Funding. Hager:Tetralogic Pharmaceuticals: Research Funding. Frey:Tetralogic Pharmaceuticals: Research Funding. Andreeff:TetraLogic Pharmaceuticals: Research Funding. Carter:Tetralogic Pharmaceuticals: Research Funding. Minderman:Tetralogic Pharmaceuticals: Research Funding. Russell:Tetralogic Pharmaceuticals: Employment. Tibes:Tetralogic Pharmaceuticals: Research Funding.