ALBERT

Description: CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies. Yet CD19 CAR T cells fail to induce durable responses in most patients. Second infusions of CD19 CAR T cells (CART2) have been considered as a possible approach to improve outcomes. We analyzed data from 44 patients with R/R B-cell malignancies (ALL, n=14; CLL, n=9; NHL, n=21) who received CART2 on a phase 1/2 trial at our institution. Despite a CART2 dose increase in 82% of patients, we observed a low incidence of severe toxicity after CART2 (grade ≥3 CRS, 9%; grade ≥3 neurotoxicity, 11%). After CART2, CR was achieved in 22% of CLL, 19% of NHL, and 21% of ALL patients. The median durations of response after CART2 in CLL, NHL, and ALL patients were 33, 6, and 4 months, respectively. Addition of fludarabine to cyclophosphamide-based lymphodepletion before CART1 and an increase in the CART2 dose compared to CART1 were independently associated with higher overall response rates and longer progression-free survival after CART2. We observed durable CAR T-cell persistence after CART2 in patients who received Cy-Flu lymphodepletion before CART1 and a higher CART2 compared to CART1 cell dose. The identification of two modifiable pre-treatment factors independently associated with better outcomes after CART2 suggests strategies to improve in vivo CAR T-cell kinetics and responses after repeat CAR T-cell infusions, and has implications for the design of trials of novel CAR T-cell products after failure of prior CAR T-cell immunotherapies.

Description: CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is a novel treatment with promising results for patients with relapsed/refractory lymphoid malignancies. CAR-T cell therapy has known early toxicities of cytokine release syndrome (CRS) and neurotoxicity, but little is known about long-term neuropsychiatric adverse effects. We have utilized patient-reported outcomes (PROs), including PROMIS®measures, to assess outcomes of patients with relapse/refractory chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and acute lymphoblastic leukemia (ALL) who were treated with CD19-targeted CAR-T cells on a clinical trial in our institution (NCT01865617) and survived at least one year after treatment. Between October 2018 to February 2019, 52 patients (at their 1-5 year anniversary after CAR-T cell therapy) were sent a questionnaire. The questionnaire included the PROMIS Scale v1.2-Global Health and the PROMIS-29 Profile v2.1, as well as 30 additional questions, including questions pertaining to cognitive function. As of February 28, 2019, 40 questionnaires were returned (76.9% response rate) and were included in the analysis. Patients' characteristics are summarized in Table 1. Cognitive function was assessed by asking if patients had experienced difficulties with concentration, finding words, memory, or solving problems since their CAR-T cell therapy; answer "yes" to each of the questions received "1" point to determine the total cognitive difficulty score (0-4). PROMIS measures are standardized to a T-score metric, with a score of 50 representing the general US population mean. Clinically meaningful differences were defined as a 5-point difference in scores (1/2 standard deviation). The cohort's self-reported cognitive difficulties and PROMIS mean T scores are shown in Table 2. Mean T scores of PROMIS domains of Global Mental health, Global Physical Health, Social Function, anxiety, depression, fatigue, pain and sleep disturbance were not clinically meaningfully different from the mean in the general US population. However, 19 participants (47.5%) reported at least one cognitive difficulty and/or clinically meaningful depression and/or anxiety (Figure 1), and 7 participants (17.5%) scored ≤ 40 in Global Mental Health, indicating at least one standard deviation worse than the general population mean. On risk factor analysis, younger age was found to be associated with worse Global Mental Health (p=0.02), anxiety (p=0.01) and depression (p=0.01). Anxiety prior to CAR-T cell therapy was associated with increased likelihood of anxiety after CAR-T cell therapy (p=0.001). Multivariate analysis confirmed association between age and PROMIS Global Mental Health score (p=0.03). 15 participants (37.5%) reported cognitive difficulties post CAR-T cell therapy. On multivariate analysis, depression prior to CAR-T cell therapy was statistically significantly associated with higher likelihood of self-reported cognitive difficulties after CAR-T therapy (p=0.02) and there was a trend for association between acute neurotoxicity after CAR-T cell infusion and self-reported long-term cognitive difficulties (p=0.08). Having more cognitive difficulties was associated with worse Global Mental Health (p=0.0001) and worse Global Physical Health (p= 0.01). Similarly, worse scores for pain interference, sleep disturbance, fatigue, depression, anxiety, physical function, and social function were associated with more long-term self-reported cognitive difficulties (p=0.007,p=0.0003, p=0.00006, p=0.01, p=0.0007, p=0.003, p=0.0004 respectively). Our study demonstrates overall good neuropsychiatric outcomes in 40 long-term survivors after CAR-T cell therapy. However, despite good overall mean scores, nearly 50% of patients in the cohort reported at least one negative neuropsychiatric outcome (anxiety, depression or cognitive difficulty), and almost 20% scored at least one standard deviation lower than the general US population mean in Global Mental Health, indicating that there is a significant number of patients who would likely benefit from mental health services following CAR-T cell therapy. Younger age, anxiety and depression pre-CAR-T cell therapy, and acute neurotoxicity after CAR-T cell infusion may be risk factors for long-term neuropsychiatric problems in this patient population. Larger studies are needed to confirm these findings. Disclosures Shaw: Therakos: Other: Speaker Engagement. Lee:AstraZeneca: Research Funding; Incyte: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Kadmon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Turtle:T-CURX: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Allogene: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Ad hoc advisory board member. Maloney:Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; A2 Biotherapeutics: Honoraria, Other: Stock options ; Celgene,Kite Pharma: Honoraria, Research Funding; BioLine RX, Gilead,Genentech,Novartis: Honoraria.

Description: Recently two CD19-targeted CAR-T cell products were approved by the FDA for treatment of relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). Excellent anti-tumor activity has been observed in patients with B cell malignancies. However, data regarding long-term effects of this therapy are very limited. Here we report long-term effects in 59 patients (pts) with R/R NHL and chronic lymphocytic leukemia (CLL) who received a total of 85 CD19-targeted CAR-T cell infusions on a clinical trial in our institution (NCT01865617), survived more than a year, and had at least one year follow-up data after their first CAR-T cell infusion. One patient who survived more than a year was excluded from this report due to incomplete data. Median follow-up was 23 months (range, 13-57) after the first CAR-T cell infusion. We report adverse events that occurred or persisted beyond 90 days after the last CAR-T cell infusion, excluding events related to disease progression. Median age at CAR-T cell infusion was 60 years (range, 34-73). There were 42 (71%) pts with NHL and 17 (29%) with CLL. The median number of prior lines of treatment was 4 (range, 1-8). 23 (39%) pts had received prior autologous (auto) hematopoietic cell transplantation (HCT), and 9 (15%) pts had received prior allogeneic (allo) HCT. 35 (59%) pts received one CAR-T cell infusion, 22 (37%) pts received 2 infusions, and 2 (3%) pts received 3 infusions. 3 (5%) pts received a maximum cell dose of 2x10(5)/kg, 40 (68%) pts received a maximum cell dose of 2x10(6)/kg, and 16 (27%) pts received a maximum cell dose of 2x10(7)/kg. 65 (76%) infusions were preceded by cyclophosphamide and fludarabine. CRS grade I/II occurred in 38 (64%) pts, and grade III in 4 (7%) pts (graded per Lee et al. Blood, 2014). No grade IV CRS was reported in this cohort. Acute neurotoxicity occurred in 20 (34%) pts. At 2 months after CAR-T cell infusion complete response (CR) was documented in 34 (58%) pts, partial response (PR) in 12 (20%) pts, and disease progression (PD) in 13 (22%) pts. During the follow-up period, another 15 (25%) pts developed PD. 29 (49%) pts received salvage therapy after CAR-T cell infusion, 8 (14%) of them received allo HCT. 5 (8%) pts received allo HCT as consolidation after CAR-T cell. 5 of 25 (20%) pts who did not receive additional therapy after last CAR-T cell infusion experienced ongoing cytopenias requiring G-CSF support, or RBC or platelet transfusions, beyond 90 days after last CAR-T cells infusion. 8 (14%) pts were diagnosed with subsequent malignancies, including 3 (5%) myelodysplasia, 4 (7%) non-melanoma skin cancer, and 1 non-invasive bladder cancer. All, but 1 patient with skin cancer, had auto or allo HCT before CAR-T cell therapy. Neuropsychiatric disorders were documented in 5 (8%) pts; including major depression, suicidal attempt, myoclonic seizures, and TIA. 5 (8%) pts experienced cardiovascular events. 4 (7%) pts developed renal dysfunction. 3 (5%) pts developed respiratory disorders. One pt had gastrointestinal bleeding. Of the 9 pts who had undergone allo HCT before CAR-T cell therapy, 1 pt (11%) developed GVHD flare. Severe hypogammaglobulinemia (IgG 〈 400 mg/dL) or IgG replacement beyond day 90 after last CAR-T cell infusion (and before HCT if was done) were documented in 24 (41%) pts. 54 pts were included in the infection analysis. 178 suspected infection events beyond day 90 after last CAR-T cell infusion were documented in 40 (74%) pts. Antimicrobial treatments were documented for 124 infection events. 44 (25%) of the events were microbiologically proven. The most common infections were upper (92) and lower (29) respiratory tract infections. 25 (46%) pts required hospital admission due to infections, of them 8 (15%) were admitted to the ICU. When excluding infections that occurred after salvage therapy following CAR-T cell, we identified 117 infections in 28 (52%) pts. 3 pts died of non-relapse causes (2 due to infection after allo HCT, and 1 due to duodenal ulcer and gut perforation). In conclusion, our data suggest that long-term effects of CD19-targeted CAR-T cell therapy are acceptable. Most effects identified in our cohort were not severe, and many may have been related to prior or subsequent therapies (e.g. HCT before or after CAR-T cell therapy, or subsequent salvage treatments). Our data is consistent with recent published data demonstrating excellent long-term disease outcome for this heavily pre-treated population. Disclosures Turtle: Juno/Celgene: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribu Biosciences: Membership on an entity's Board of Directors or advisory committees. Maloney:Juno Therapeutics: Research Funding; GlaxoSmithKline: Research Funding; Janssen Scientific Affairs: Honoraria; Roche/Genentech: Honoraria; Seattle Genetics: Honoraria.

Description: Background Chimeric antigen receptor therapy (CAR-T) directed against CD19 has demonstrated efficacy in patients with relapsed/refractory (R/R) B-cell malignancies. Delayed hematopoietic recovery with grade 3/4 neutropenia and thrombocytopenia, requiring extended growth factor administration or transfusions, has been observed in patients undergoing CAR-T cell therapy, although the factors influencing recovery are poorly understood. In this study, we performed multivariable analyses to identify factors associated with hematopoietic recovery in patients undergoing CD19 CAR-T cell therapy. Methods We retrospectively analyzed 125 patients with R/R acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia (CLL), treated with CD19-targeted CAR-T cells on a phase 1/2 clinical trial in our institution (NCT01865617). Patients receiving more than one CAR-T infusion were excluded. Criteria for neutropenia, thrombocytopenia, and recovery were defined as per the Center for International Blood and Marrow Transplant Research (CIBMTR) reporting guidelines: neutropenia, absolute neutrophil count (ANC) ≤ 500/mm3; thrombocytopenia, platelet (Plt) count ≤ 20 x 109/L; neutrophil recovery, ANC 〉 0.5 x 109/L for three consecutive laboratory values obtained on different days irrespective of growth factor administration; platelet recovery, Plt 〉 20 x 109/L for three consecutive values obtained on different days in the absence of platelet transfusion for seven days. For competing risk analysis, an event was defined as having achieved ANC or Plt recovery, with the following considered as competing events: death, new cytotoxic therapy, relapse with marrow involvement in the absence of ANC or platelet recovery. Patients who never met the CIBMTR criteria for neutropenia of thrombocytopenia were considered as having recovered at time = 0. To identify factors associated with impaired hematopoietic recovery after CD19 CAR-T cell therapy, patient-, disease- and CAR-T cell therapy-related variables were included in a multivariable Fine and Gray model prior to variable selection using LASSO penalization (Table 2 footnote). Results We included 125 patients (ALL, n=44; CLL, n=37; NHL, n=44) with a median age of 55 (range, 20-76). Patients were heavily pre-treated with a median of 4 prior therapies (range, 1-10); 31% had undergone prior autologous or allogeneic hematopoietic cell transplantation (HCT). Median ANC and Plt prior to lymphodepletion were 2 x 109/L (range 0-23) and 112 x 109/L, range 3-425), respectively. Patient and treatment characteristics are summarized in Table 1. ANC and Plt recovery after CD19 CAR-T cell therapy were observed in 91% (ALL, 86%; CLL, 92%; NHL, 95%) and 86% (ALL, 86%; CLL, 86%; NHL, 84%) of patients, respectively. Median time to ANC recovery was 9 days and the probability of ANC recovery at day 28, 60, and 90 was 80% (95%CI, 73-87), 86% (95%CI, 80-92) and 89% (95%CI, 83-94), respectively. The probability of platelet recovery on the day of CAR-T cell infusion was 55% (95%CI, 46-64); rising to 74% (95%CI, 67-82), 83% (95%CI, 76-90), and 84% (95%CI, 77-90) at day 28, 60, and 90, respectively. A competing event was always observed in patients without ANC or Plt recovery. In multivariable analysis, higher pre-lymphodepletion Plt count (HR=1.08 per 25 x 109/L increase, p=0.006) and higher peak CD8+ CAR-T cells in blood (HR=1.47 per log10 cells/µL increase, p

Description: Abstract 354 Relapse is the leading cause of death after allogeneic hematopoietic cell transplantation (HCT) for hematological malignancies. Relapse after HCT can be treated with donor lymphocyte infusion (DLI). Initial low dose DLI followed by escalating DLI doses has been used to minimize the risk of GVHD in patients with relapsed chronic myeloid leukemia. The impact of initial CD3+ cell dose on outcome after DLI in patients treated for other hematological malignancies is limited. We conducted a retrospective analysis of 225 patients with relapsed hematological malignancies after HCT treated with DLI over the last 20 years. The primary objective of this study was to determine the effect of the initial DLI CD3+ cell dose on graft-versus-host disease (GVHD) and overall survival (OS). Other potential risk factors for the development of GVHD (i.e. patient/donor gender, patient age, HLA-match, diagnosis, conditioning type, prior acute or chronic GVHD, donor chimerism, lymphocyte count, interval between transplant to DLI, and initial DLI TNC dose) and for survival after DLI (i.e. diagnosis, disease status at DLI, transplant conditioning type, time from transplant to relapse and to DLI, prior history of GVHD) were included in the analysis. For assessment of survival, patients were classified into a high risk group (AML, MDS, ALL, CML (BC, AP), high grade lymphomas (Hodgkin lymphoma, DLBCL, transformed NHL)) (n=145) or low risk group (CML-CP, CLL, MM, other lymphomas) (n=80). The median age of the cohort was 46 (range, 3–74) and 59% (n=132) were male. DLI was given for treatment of chronic myeloid leukemia (n=56), acute myeloid leukemia (n=71), myelodysplastic syndrome (n=25), acute lymphoblastic leukemia (n=21), multiple myeloma (n=23), lymphoma (n=21) and chronic lymphocytic leukemia/lymphoma (n=11). Patients received transplants from HLA-matched related (n=171) or unrelated (n=41) donors. Thirteen patients had HLA-mismatched donors. 58 patients (26%) received non-myeloablative conditioning regimens. The median time intervals were 11.3 months (range, 0–180) from HCT to relapse and 15.5 months (range, 21.1–215) from HCT to DLI. 144 patients (64%) received cytoreductive therapy before DLI, and 55 patients (24%) had complete remission at time of DLI. Initial DLI CD3+ cell dose/kg was ≤1×107 (n=84; Group A), 〉1.0 to

Description: Abstract 350 Relapse is the leading cause of death following allogeneic hematopoietic cell transplant (HCT) for hematological malignancies. Although evidence suggests that the beneficial donor T cell-mediated graft versus leukemia (GVL) effect can reduce post-HCT relapse rates, this is often mitigated by morbidity and mortality associated with the accompanying graft versus host disease (GVHD). Thus, providing antigen-specific T cells that selectively target leukemia associated antigen (LAA) constitutes a distinct opportunity to promote GVL activity without inducing GVHD. Wilms' Tumor Antigen 1 (WT1) is a non-polymorphic zinc finger transcription factor that plays a key role in cell growth and differentiation. WT1 has a very limited expression in normal tissues, is expressed 10–1000× fold more in leukemia cells compared to normal CD34+ cells, and has been shown to contribute to leukemogenesis. WT1 is expressed in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and acute lymphoid leukemia (ALL). Furthermore, the magnitude of expression of WT1 in leukemic cells correlates with prognosis and clinical aggressiveness. Thus, WT1 constitutes an attractive candidate target for CD8+cytotoxic T-cells (CTL) (Cheever et al. Clin Cancer Res 2009;15(17):5323–5337). In this study, escalating doses of clonal populations of donor-derived CD8+ CTL specific for the HLA A*02:01-restricted WT1126–134 (RMFPNAPYL) epitope were administered to 11 high-risk leukemia patients after allogeneic HCT. The absence of end-organ toxicities or the development of new-onset GVHD demonstrated that the infusions were safe and well-tolerated. As the persistence of transferred cells was limited in some patients, the last four patients received CTL clones primed in the presence of the γc-chain cytokine Interleukin-21 (IL-21), a culture strategy recently shown to confer a less differentiated phenotype to T cells generated in vitro, as a means to increase the ability of transferred cells to survive in vivo and potentially mediate greater anti-leukemic activity (Li, Y et al. J Immunol 2005;175:2261–2269). Four patients, who were treated not in florid relapse (3 in CR and 1 with MRD entering infusions) but were at high risk for relapse post-HCT (40–55% relapse rate at one year post HCT), and received CTL generated in the presence of IL-21 have survived for 22 to 37 months post-HCT without detectable leukemia or relapse, and in the absence of additional anti-leukemic treatment or GVHD (Table 1). In these four patients, transferred CTL remained detectable for 8 to 15 month after T cell infusions (Fig. 1), and maintained/upregulated in vivo phenotypic (CD27, CD28, CD127, CD62L and CCR7) and functional (the ability to produce IL-2 in response to cognate antigen) characteristics associated with long-lived memory CD8 T-cells (Fig. 2). Direct evidence of transient anti-leukemic activity was observed in one patient treated with advanced progressive disease, and of a prolonged response in a patient with minimal residual disease. The results of this study suggest that transfer of donor-derived WT1-specific CTL clones can be accomplished without significant toxicity and can potentially provide therapeutic anti-leukemic activity. Table 1. Clinical Outcomes Figure 1. In vivo persistence of WT1-specific CTL clones and effect on leukemia disease burden Figure 1. In vivo persistence of WT1-specific CTL clones and effect on leukemia disease burden Figure 2. Adoptively transferred WT1-specific CD8+T-cells persisting in vivo exhibit many phenotypic and functional characteristics associated with CD8+central memory cells Figure 2. Adoptively transferred WT1-specific CD8+T-cells persisting in vivo exhibit many phenotypic and functional characteristics associated with CD8+central memory cells Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Allogeneic Hematopoietic Cell Transplantation (allo HCT) is currently the only curative therapy for high-risk hematologic malignancies due to the immune response of the donor cells against the malignant cells (graft versus tumor effect; GVT), but with the cost of Graft Versus Host Disease (GVHD). Despite extensive research, very few predictors of GVHD and GVT have been identified to date. Additionally, clinical GVHD diagnosis can be challenging due to chemotherapy-related or infection-related organ toxicity manifestations, which further complicate prediction and treatment stratification algorithms. In order to study the mechanisms of GVHD and GVT and to identify potential GVHD markers we apply a novel approach, called Transcriptome Fingerprint Assay (TFA), relying on high frequency sampling and blood transcript profiling. The TFA is a multiplex microfluidics q-PCR based assay linked with a computational model for modular functional transcriptome analyses, uniquely tailored to answer complex questions on immune perturbations through frequent profiling of gene expression signatures from 〈 1 ml of blood (Chaussabel and Baldwin. Nat Rev Immunol 2014, Speake et al. Clin Exp Immunol 2017). This approach has been successfully applied to stratify patients' prognosis in autoimmune and infectious diseases (Banchereau R et al. Cell 2016, Dunning et al. Nat Immunol 2018). In our study we use the TFA to capture longitudinal immune signatures as dynamic "snapshots" of the patient's immune system after HCT. Hypotheses Fluctuations over-time in gene expression of allo HCT patients' immune system reflect the pathologic/disease control programs (GVHD/GVT) and may be used to identify diagnostic and predictive biomarkers. GVHD/GVT control immune programs depend on the "inner" interface between the donor immune-system and the recipient, and are influenced by external variables, as infections or drugs. These variables can affect the immune system-related gene expression and can be measured. Objectives To systematically measure gene expression signatures in immune perturbations post-allo HCT, in order to: Identify GVHD-related immune signatures consistent with clinical diagnosis of GVHD.Predict and stratify therapy-resistant GVHD and severe chronic GVHD, according to immune signatures.Identify links (causative and consequential) between GVHD, GVT, relapse, and other post-transplant immune perturbations (e.g. infections). Methods Enroll 250 allo HCT patients to populate a "GVHD cohort" and a "non-GVHD cohort" of 50 patients each, and 50 donors (healthy controls cohort) . Patients donate micro-quantities of blood (50 to 600 microliters), weekly until day 100 post-transplant and every 2 weeks thereafter until 2 years after transplant. Detailed clinical, laboratory and therapy annotations are captured during the follow-up. Gene expression of 264 immune-related genes for each sample are measured through Fluidigm BioMark high throughput qPCR system, and normalized to the geometric mean Ct of 8 housekeeping genes. Data interpretation is performed through TFA modular analyses and correlated with the clinical annotations. Results Results of three series of patients' samples are shown to exemplify the potential of TFA as a method to study the mechanisms of GVHD and GVT. All three patients underwent myeloablative peripheral blood stem cell transplant from an HLA identical sibling donor. Two patients developed steroid responsive-acute GVHD (patient #1: GVHD stage I was diagnosed on day 38 post HCT, patient #4: GVHD stage III was diagnosed on day 21 post HCT). One patient (Patient #6) did not develop clinical GVHD, but routine skin biopsy on day 80 revealed apoptotic cells consistent with subclinical skin GVHD. Principal Component Analysis (PCA) of the three patients' series is shown in Figure 1, the dynamic transcriptomes according to TFA modules of patients #1 and #6 are shown in Figure 2, and representative TFA modular fingerprints are shown in Figure 3. Conclusion We anticipate that using the TFA approach will help to fill knowledge gaps instrumental to solve clinical dilemmas related to allo HCT complications, and to improve the clinical outcomes of allo HCT patients. Disclosures No relevant conflicts of interest to declare.