CD19-negative Relapse Research Articles

Changes in Leukemic Blasts CD19 Expression in Children with Relapsed/Refractory B-Cell Precursor ALL Treated with Blinatumomab

Blinatumomab is a bispecific T-cell engager (BiTE) antibody construct targeting CD3 and CD19, resulting in T-cell-mediated lysis of malignant B cells. Currently the main application of blinatumomab is the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although promising results were obtained therapy with blinatumomab fails in 30-60% of patients. CD19 downexpression is shown to be one of the possible mechanisms of tumor cells adaptation and subsequent escape from treatment. Moreover, loss of CD19 is a serious obstacle for flow cytometric minimal residual disease (MRD) assessment, necessary for control the leukemia response to therapy. The aim of the present study was to evaluate changes of CD19 expression in children with BCP-ALL treated with blinatumomab and possible monitoring of MRD using additional markers.Patients and methods. From December 2015 to July 201739 (18 F/21M) patients with median age of 8 years (8 mo-20 y) with relapsed/refractory (R/R) BCP-ALL completed at least one course of blinatumomab. 5 patients (13%) carried various types of KMT2A-gene rearrangements and in 3 (7,5%) children ETV6-RUNX1 fusion transcript was detected. MRD monitoring was performed by 8-color flow cytometry. After CD19-directed treatment CD22 and CD24 were added to conventional antibodies' combinations for MRD detection (as suggested by Cherian S. et al, Cytometry B. 2016) to avoid negative influence of potential CD19 loss on cytometric data analysis and interpretation. MRD-negativity was defined as undetectable at the sensitivity level of 0.01%.Results. 20 out of 39(51%) patients continue in complete continuous remission(CCR) after one or two blinatumomab courses, followed by alloHSCT.In 8 (40%) patients in CCR, MRD was never detected since completion of first blinatumomab course, and they were excluded from further analysis. In other 12 pts were found to be MRD-positive at least once after blinatumomab administration. Among them in 5 patients leukemic blasts were still CD19-positive and in 7 patients residual leukemic cells were CD19-negative.Among 19 out of 39 patients (49%) who were resistant to(n=7) or developed bone marrow relapse after blinatumomab (n=12), in 12 (63%) cases tumor cells at relapse were CD19-positive, whereas 5 children experienced CD19-negative relapse. Among them only CD19-positive MRD was detected in one case, only CD19-negative residual blasts - in two cases and in other two children both types of CD19 expression were found atdifferent time-points.Two additional patients, who had CD19-negative MRD, then developed relapse as a lineage “switch” from BCP-ALL to acute myeloid leukemia (AML). In these patients myeloid switch developed in a different ways: one patient carried KMT2A-AF4 fusion gene which is more or less typical for switching AL, the second patient with germline KMT2A hadvery small AML population at the time of relapse diagnosis. Thus in this case positive selection of myeloid subclone during the blinatumomab treatment could be the main pathway of AML development.Conclusions. In our group the remission rate after blinatumomab treatment was about 50%. In those children, who didn't respond and/or developed subsequent relapse, CD19 downexpression seems to be one of the significant pathways for tumor escape from blinatumomab - 26% of relapses were CD19-negative, and in 42% of all relapsed/resistant casesCD19-negative blasts were detected at least on MRD level. Rather frequent loss of CD19 could be a serious obstacle for flow cytometric MRD monitoring, but application of expanded antibodies panel and use of 10-12-color investigation allows residual blasts detection in nearly all cases. Other mechanisms of leukemia resistance including myeloid switch (even in KMT2A-germline patients) can cause blinatumomab failure. DisclosuresNo relevant conflicts of interest to declare.

Updated outcomes with axicabtagene ciloleucel (axi-cel) retreatment (reTx) in patients (pts) with relapsed/refractory (R/R) indolent non-Hodgkin lymphoma (iNHL) in ZUMA-5.

7548 Background: ZUMA-5 is a Phase 2 study of axi-cel anti-CD19 CAR T-cell therapy in pts with R/R iNHL (follicular lymphoma [FL]; marginal zone lymphoma [MZL]). In the primary analysis, 11 pts (9 FL; 2 MZL) were retreated with axi-cel, achieving an overall response rate (ORR) of 100% (91% complete response [CR] rate) at a median follow-up of 2.3 mo post-reTx, with no Grade ≥3 cytokine release syndrome (CRS) or neurologic events (NEs; Chavez et al. ASH 2020. #2036). Here, we report updated clinical and translational outcomes with longer follow-up in pts retreated with axi-cel in ZUMA-5. Methods: Eligible pts with FL or MZL had R/R disease after ≥2 lines of therapy. Pts were considered for reTx if they progressed after a response at mo 3, had no evidence of CD19-negative relapse in biopsy, had no axi-cel neutralizing antibodies, and had no Grade 4 CRS or NEs with 1st Tx. Retreatment was per investigator discretion. At both Txs, pts received axi-cel (2×106 CAR T cells/kg) after conditioning chemotherapy. Results: As of 9/14/2020, 13 pts with iNHL (11 FL; 2 MZL) received axi-cel reTx, with 2 pts retreated after the primary analysis. Before their 1st Tx, pts had median 4 prior lines of therapy; 85% had stage 3–4 disease; 82% had FLIPI of ≥3; 46% were POD24; 77% had refractory disease. Among the 13 retreated pts, 85% had a CR to 1st Tx. Median 1st duration of response (DOR) was 8.2 mo. Detectable CD19 was confirmed in all evaluable biopsies from retreated pts at relapse, and median time from 1st Tx to reTx was 10.6 mo. Following reTx, the ORR was 100% (77% CR rate). After a median follow-up of 11.4 mo, the median DOR had not yet been reached; 46% of retreated pts had ongoing responses at data cutoff. At 1st Tx, CRS occurred in 9 pts (5 Grade 1, 4 Grade 2); NEs occurred in 5 (3 Grade 1, 1 Grade 2, 1 Grade 3). At reTx, CRS occurred in 8 pts (6 Grade 1, 2 Grade 2); NEs occurred in 4 (3 Grade 1, 1 Grade 2). Median peak levels of biomarkers typically associated with severe CRS and NEs were similar at reTx and 1st Tx (IL-6, 7.7 vs 5.7 pg/mL; IL-2, 1.8 vs 0.9 pg/mL; IFN-γ, 62.9 vs 64.2 pg/mL). In the 11 retreated pts with FL, tumor burden (median sum of product diameters [SPD]) was lower before reTx vs 1st Tx (1416 vs 4770 mm2). Engraftment index (CAR T-cell expansion relative to SPD) is an indirect proxy for effector:target ratio and a key covariate of response to axi-cel (Locke et al. Blood Adv. 2020). Though median peak CAR T-cell levels appeared lower at reTx vs 1st Tx (5.2 vs 14.3 CAR+ cells/µL blood), engraftment index was similar (0.003 vs 0.005 cells/µL×mm2). Conclusions: Axi-cel reTx achieved deep and durable responses, with an acceptable safety profile. Tumor CD19 positivity was maintained at relapse, and engraftment index was similar at both Txs, comparing favorably to previous reports in aggressive lymphomas (Locke et al. ASCO 2020. #8012). These data suggest axi-cel reTx is a promising option for pts with R/R iNHL. Clinical trial information: NCT03105336.

Determinants of CD19-positive vs CD19-negative relapse after tisagenlecleucel for B-cell acute lymphoblastic leukemia.

Tisagenlecleucel therapy has shown promising efficacy for relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, relapses occur in 30-50% of patients. Determinants for CD19pos versus CD19neg relapses are poorly characterized. We report on 51 patients with R/R BCP-ALL (median age 17 years) infused with tisagenlecleucel after lymphodepletion. Complete remission rate at D28 was 96%. Prior blinatumomab increased the risk of early failure at D28. The 18-month cumulative incidence of relapse (CIR), event-free survival (EFS), and overall survival (OS) were 51%, 44%, and 74%, respectively, at a median follow-up of 15.5 months. Factors associated with a high tumor burden (occurrence of cytokine release syndrome) and prior blinatumomab were associated with an increased CIR, and a shorter EFS and OS. Pre-lymphodepletion high disease burden (MRD ≥ 10-2, SHR 10.4, p = 0.03) and detectable MRD at D28 (SHR 7.2, p = 0.006) correlated with an increased risk of CD19neg relapse. Low disease burden (SHR 5.3, p = 0.03) and loss of B-cell aplasia (BCA) (SHR 21.7, p = 0.004) predicted an increased risk of CD19pos relapses. These data highlight the impact of prior therapy on patient outcome. Finally, detectable MRD at D28 and loss of BCA both define patients at high risk of relapse for whom additional interventions are needed.

Genetic Mechanism of Leukemia Relapse Following CD19 Chimeric Antigen Receptor T Cell Therapy.

Chimeric antigen receptor T cell therapy (CART) has achieved excellent results in the past 10 years for treating of leukemia. Treatment of B cell acute lymphoblastic leukemia by anti-CD19 CART can reach a complete remission rate of 90%. Although CART has greatly improved the treatment of patients with leukemia and lymphoma, as many as one-third of the patients can suffer disease relapse after CART. The tumor surface marker CD19 is negative in most the patients who relapse, and these patients display high expression of CD19 before treatment. In this review, the current causes of CD19-negative relapses after CD19 CART against leukemia, and the mechanisms of target escape are briefly summarized. Also, methods and strategies for treating relapse to provide references for the treatment of leukemia relapse are also discussed.

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

Ligand Based CAR T-Cell Targeting BAFF Receptors Asa Novel Therapy for B Cell Malignancies

Background: Autologous T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have shown rapid and durable responses in B cell malignancies. Although CD19 CAR-T cells have demonstrated remarkable success, CD19-negative relapses occur in 30-45% of patients, highlighting the need for adoptive immunotherapies with alternative targeting approaches. B-cell activating factor (BAFF) is a critical B cell survival factor. Receptors of BAFF (BAFF-R, TACI and BCMA) are expressed by a wide range of B cell neoplasms, including ALL, CLL, NHL and MM, making them attractive therapeutic targets. We developed a novel ligand-based CAR that when expressed in T cells, targets and eliminates malignant B cells expressing BAFF receptors (BAFF CAR-T). This approach has several potential advantages over CD19 targeting CAR-T therapy: CD19 is expressed on all B cells, but BAFF receptors are expressed only on mature B cells, making it a more specific antigen for targeting and potentially narrowing down the side effect profile. BAFF CAR-T cells are a potential therapeutic strategy to treat CD19 CAR-T relapsed patients as well as chemotherapy resistant patients. Methods: BAFF ligand was fused to a second generation CAR backbone containing 4-1BB costimulatory and CD3ζ intracellular signaling domains. T cells were isolated from human blood, activated and transduced with BAFF-CAR lentiviral particles. In vitro tumor cell killing was analyzed using calcein-AM cytotoxicity assay. For in vivo testing of BAFF CAR-T cytotoxicity, we used mantle cell lymphoma (MCL) Jeko-1 xenograft model. Immunocompromised NSG mice were subcutaneously injected with human MCL cell line Jeko-1 (10.106 cells at day 0). Once these mice developed measurable tumors, we injected T cells transduced with empty vector (control T cells) or BAFF-CAR T cells (10 x 106 cells) or PBS intra-tumorally as a one-time injection. Tumor volumes were measured every other day using calipers. Results: BAFF CAR-T cells showed significant cytotoxicity in vitro (not shown) and in vivo against human MCL cell line Jeko-1. Mice treated with BAFF-CAR-T showed significant reduction in tumor volume compared to mice treated with control T cells and PBS (Figure 1A, B). Tumor progression was observed after control T cell and PBS treatment, whereas the cohort treated with BAFF CAR-T did not show any tumor progression, and with complete or near-complete tumor eradication. Survival analysis showed the BAFF CAR-T treated cohort had significantly longer survival compared to control-T cell and PBS treated cohorts (Figure 1C). Mice were sacrificed when tumor volume reached 2 cm3. Conclusion: Our data suggest that targeting BAFF receptors with a novel, ligand-based BAFF-CAR-T is a feasible and effective immunotherapeutic strategy to eliminate malignant B cells, warranting further development. BAFF-CAR-T cells have therapeutic potential against a wide spectrum of B cell malignancies, including CD19 negative relapsed disease. Clinical grade expansion and clinical trials are in development for BAFF CAR-T therapy non Hodgkin lymphoma patients. Disclosures Parameswaran: Luminary Therapeutics: Consultancy; Luminary therapeutics: Research Funding. de Lima:Kadmon: Other: Personal Fees, Advisory board; BMS: Other: Personal Fees, advisory board; Incyte: Other: Personal Fees, advisory board; Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board; ADC therapeutics: Other: Advisory Board, Research Funding; Celgene: Speakers Bureau.

Clinical Efficacy of Polatuzumab Vedotin in Patients with Relapsed/Refractory Large B-Cell Lymphoma after Standard of Care Axicabtagene Ciloleucel

Introduction. The outcome of patients with large B-cell lymphoma (LBCL) relapsing or progressing after anti-CD19 CAR T-cell therapy is dismal, and novel therapeutic strategies are needed. Polatuzumab vedotin (PV) is a CD79b-directed antibody-drug conjugate, approved by the FDA in combination with bendamustine and rituximab for the treatment of patients with LBCL who relapse or progress after at least 2 lines of systemic therapy. CD79b targeting is an appealing strategy after failure of anti-CD19 CAR T-cell therapy, but the activity of PV in this setting has not been investigated. Methods. This is a retrospective analysis of patients with relapsed/refractory LBCL after standard of care axicabtagene ciloleucel (axi-cel), and treated with standard of care PV at MD Anderson Cancer Center between 07/2019 and 03/2020. PV was given at the standard dose of 1.8 mg/kg IV every 3 weeks in all patients. PV was administered with rituximab, with or without bendamustine. Response to treatment and progression were defined according to 2014 Lugano criteria. Results. Eight patients were included in the analysis: median age was 54 (range, 41-70 years), 7 (87.5%) were male, and all had an IPI score > 3. Median number of systemic therapies before axi-cel was 3 (range, 2-7), and median number of systemic therapies between axi-cel and PV use was 1 (range, 0-3); 1 patient previously had autologous stem cell transplant (SCT), and 2 had allogeneic SCT. Three (37.5%) patients were primary refractory to axi-cel therapy, and median time from axi-cel to PV was 6 months (range, 1-12 months). All patients were biopsied at time of relapse after axi-cel, and in all cases lymphoma was CD19+ CD79b+ by immunohistochemistry/flow cytometry. At time of PV initiation, median absolute neutrophil count was 2.6 (range, 0.4-4.7 X109/L), median platelet count was 141 X109/L (range, 7-245 X109/L), median serum creatinine was 0.9 mg/dL (range, 0.5-1.5 mg/dL), and all patients had LDH above upper limit of normal. Median number of PV cycles was 2 (range, 1-3): PV was combined with rituximab in all patients but administered with bendamustine in only 3 (37.5%). No significant toxicity, prompting dose reduction or treatment discontinuation, was observed. A response was achieved in 4 (50%) patients, represented by partial remission (PR) in all cases, whereas 4 (50%) patients were refractory. All patients stopped PV, 6 (75%) because of progression, 1 (12.5%) to proceed to allogeneic SCT while in PR, and 1 (12.5%) to proceed to an immunotherapy clinical trial (despite absence of progression). After a median follow up of 29 weeks (95% CI, 16-31 weeks), all patients progressed/died, and median PFS was 5 weeks (95% CI, 2-8 weeks) (Figure). At most recent follow-up, 5 (62.5%) died, and median OS was 15 weeks (95% CI, 9-21 weeks)(Figure); causes of death included progression in 4 patients, and transplant-related complications in 1. Discussion. PV in combination with rituximab is safe but has limited and short-lasting activity in relapsed/refractory LBCL after anti-CD19 CAR T-cell therapy. These findings need to be confirmed in larger and prospective studies. The activity of PV, alone or in combination with novel drugs, for the treatment of patients with CD19-negative relapses after CAR T-cell therapy remains to be investigated. Figure Disclosures Westin: 47 Inc:Consultancy;Curis:Consultancy;Janssen:Consultancy;Novartis:Consultancy;Genentech:Consultancy;Juno:Consultancy;Kite:Consultancy;MorphoSys:Consultancy;Unum:Consultancy.Neelapu:Acerta:Research Funding;Takeda Pharmaceuticals:Patents & Royalties;Pfizer:Other: personal fees;Unum Therapeutics:Other, Research Funding;N/A:Other;Novartis:Other: personal fees;Adicet Bio:Other;Legend Biotech:Other;Bristol-Myers Squibb:Other: personal fees, Research Funding;Merck:Other: personal fees, Research Funding;Kite, a Gilead Company:Other: personal fees, Research Funding;Incyte:Other: personal fees;Calibr:Other;Precision Biosciences:Other: personal fees, Research Funding;Allogene Therapeutics:Other: personal fees, Research Funding;Cell Medica/Kuur:Other: personal fees;Poseida:Research Funding;Celgene:Other: personal fees, Research Funding;Cellectis:Research Funding;Karus Therapeutics:Research Funding.

CD19 CAR-T Cells Treatment Conferred a Sustained Remission in Patients with Chemotherapy-Refractory MRD in B-ALL

Background: The persistence or recurrence of minimal residual disease (MRD) after chemotherapy predicts relapse of B-cell acute lymphoblastic leukemia (B-ALL). CD19-directed chimeric antigen receptor-modified T (CD19 CAR-T) cells have shown excitable response in B-ALL. Recently, some studies have shown that ALL patients with lower burden had higher CR rate and lower risk of CRS after CAR-T therapy (Park, et al, NEJM. 2018; 378(5):439-448. Lee, et al, Blood.128 (22):Abstract 218.). However, its role in chemotherapy-refractory MRD-positive B-ALL remains unclear. Here we aimed to assess the effectiveness and safety of CD19 CAR-T in MRD-positive B-ALL patients. Methods: Since January 2018, a total of 14 B-ALL patients with persistent (n=8) or recurrent (n=6) MRD were enrolled in the CAR-T clinical trials (ChiCTR-ONN- 16009862 and ChiCTR1800015164). The patients were from two different clinical trials about CAR-T. If patients were treated in an MRD positive state, they would be included in this analysis. All the patients received one or more infusions of autogenous CD19 CAR-T. Results: Median age was 37.5 (13-62) years and 7 patients were female. The median dose of infused CAR-T cells was 6.78´106cells/kg, and 5 patients received more than one infusion. After one cycle of CAR-T infusion, 12 patients achieved MRD-negative remission, leading a response rate of 85.7%. Of the subgroup of 5 Ph-positive patients who subsequently underwent transplantation, 2 patients died due to transplant-related toxic effects, whereas the other 3 patients all currently alive without leukemia. Of the subgroup of 9 Ph-negative patients, 8 patients did not undergo subsequent transplantation (Figure 1). Three patients finally suffered CD19-positive relapse and 1 patient suffered CD19-negative relapse. Importantly, 4 patients (50%) are in ongoing molecular remission without transplantation, with a duration of response averaging 22.9 months (range: 12.1-28.6 months). The most frequent adverse events were fever and hematopoietic toxicities. Ten patients (71.4%) had grade 1 or 2 cytokine release syndrome (CRS) and no patients died of CRS. At a median follow-up time of 599.5 days (range: 172-915 days), the probability of 2-year relapse-free survival and 2-year overall survival was 61.2%±14.0% and 78.6%±11.0%, respectively. Conclusion: In conclusion, pre-emptive CD19 CAR-T treatment is an effective and safe approach and may confer a sustained remission in B-ALL patients with chemotherapy-refractory MRD. Disclosures No relevant conflicts of interest to declare.

Insights from the Greek experience of the use of Blinatumomab in pediatric relapsed and refractory acute lymphoblastic leukemia patients.

Pediatric refractory or relapsed acute lymphoblastic leukemia (ALL) poses unique therapeutic challenges, with novel immunotherapy approaches offering potential cure opportunities. In this frame, the use of Blinatumomab may induce durable remissions, serving as a successful bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT). Herein, we retrospectively summarize the Greek experience on pediatric relapsed/refractory B-cell precursor ALL patients that were treated with Blinatumomab in a compassionate, off-label setting as an effort to achieve disease clearance and proceed to allo-HSCT. In our cohort of 9 patients, 6/9 (66.7%) responded to Blinatumomab, achieving complete morphological remission (CR) after the 1st cycle, while minimal/measurable residual disease (MRD)-negativity (<10-4) after the 1st cycle was achieved in 2/2 patients (100.0%) with prior CR. A successful bridge to HSCT was feasible in 5/9 patients (55.6%). Median relapse-free survival (RFS) was 3.0 months (range 0.5-21.4 months) and median overall survival (OS) was 8.7 months (range 1.4-47.1 months) for the whole pediatric cohort. There was a trend of prolonged survival among patients who achieved MRD response after the 1st Blinatumomab administration. MRD response (defined as the >=2-log reduction of MRD value before and after Blinatumomab administration), was associated with a median RFS/OS of 7.4/7.6 months, while lack of MRD response was associated with a median RFS/OS of 0.5/3.0 months, respectively. Novel therapeutic maneuvers, in order to overcome disease resistance, i.e. increased usage of Blinatumomab dose (45 μg/m2/day), combination with donor lymphocyte infusions (DLIs), use of other immunotherapy salvage approaches (inotuzumabozogamicin), are herein discussed. Additionally, the optimal number of Blinatumomab cycles, the CD19-negative relapses and lineage switch, are also addressed. Our data although referred to a limited, however refractory or relapsed and heavily pretreated number of patients, strongly suggest that Blinatumomab may well induce sustained remissions and serve as an effective bridge to HSCT. Whether immunotherapy combined with chemotherapy can outweigh the need for subsequent allo-HSCT, if incorporated into frontline high-risk ALL therapy, remains an optimistic issue to be verified in future randomized clinical trials.

Exploring the Dilemma of Allogeneic Hematopoietic Cell Transplantation after Chimeric Antigen Receptor T Cell Therapy: To Transplant or Not?

Patients with refractory or relapsed (R/R) B cell acute lymphoblastic leukemia (B-ALL) and highly aggressive B cell non-Hodgkin lymphoma (B-NHL) have a very dismal prognosis and limited treatment options. The advent of chimeric antigen receptor (CAR) T cell therapy constitutes a milestone in current cell and gene therapies, covering the unmet need of treatment of high-risk patients and bringing immunotherapies one step closer toward cancer therapeutics, including hematologic malignancies. CAR T cells targeting CD19 antigen have shown startling remission rates in heavily pretreated B-ALL and B-NHL patients, in whom CAR T cell therapy may sometimes be their last-resort treatment. However, a high proportion of these patients evade immune surveillance by CAR T cells losing their initial deep responses, which leads to disease recurrence as either CD19-positive or CD19-negative relapse. As a result, many investigators have questioned the need for consolidative allogeneic hematopoietic stem cell transplantation (allo-HCT) after CAR T cell therapy, once a patient has achieved remission. There remains much controversy regarding whether CAR T cells should be a bridge therapy to allo-HCT or a definitive treatment, owing to the paucity of strong evidence-based data. In this context, here we review the existing data regarding the necessity, safety, and outcomes of allo-HCT performed after autologous anti-CD19 CAR T cell therapy in B-ALL and B-NHL patients.

Safety and Efficacy of Tisagenlecleucel (CTL019) in B-Cell Acute Lymphoblastic Leukemia in Children, Adolescents and Young Adults: The French Experience

Objectives: Tisagenlecleucel (CTL019) is a chimeric antigen receptor T cell- therapy that reprograms autologous T cells to target CD19+ leukemia cells, approved in the US (2017) and in the EU (2018). This study reports the feasibility, safety and efficacy of CTL019 in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) treated in Robert-Debré and Saint-Louis University Hospitals (Assistance Publique-Hôpitaux de Paris/Université de Paris). Methods: Patients (pts) with an apheresis performed between march 1, 2016 and june 15, 2019, included in sponsored-clinical trials or treated within the French compassionate program or with the commercial product, were analyzed. All infused pts received a fludarabine-cyclophosphamide based lymphodepletion before a single infusion of CAR-T cells (2 to 5 x 106 CTL019/kg if less than 50 kg; a fixed dose of 1 to 2.5 x 108 CTL019/kg if &gt; 50 kg) Results: 55 pts were referred from 25 French centers. Forty-one pts with a median number of relapses of 2 (range, 1-5) were infused with CTL019 at a median age of 18.2 y (range, 1-29.2). Eight pts were not infused due to progressive disease (n=4), screen failure (n=3) or fatal septic shock (n=1). Six pts were waiting to be infused at time of analysis. Out of the 41 infused pts, 26 had a prior history of allogeneic SCT (63%), 11 had received blinatumomab (27%). Among the 40 pts evaluable at one month post-infusion, 38 were in CR/CRi (95%) (one progression at day 5 after infusion and one toxic death at D6), 35/38 (92.1%) being clone-specific Ig-TCR MRD negative. After 3 months 21 out of 26 evaluable pts (81%) had a negative MRD. The 5 remaining MRD positive pts did relapse. No pt underwent allogeneic HSCT while in CR after CTL019 infusion. Median event free survival (EFS) and overall survival (OS) were not reached with a median follow up of 7.2 months (range, 0.2-36.3). The 18-month OS probability was 80% (95%CI, 58%-92%). The 18-month EFS probability was 58 % (95%CI, 37%-74%). Ten pts relapsed after a median time of 3.4 months (range, 1.9-10): 3 relapses were CD19+ and 5 CD19- (4 out of these 5 pts had a preexposure to blinatumomab), 2 being of undetermined status. Loss of B-cell aplasia (BCA) occurred in 9 pts after a median time of 3 months (range, 2-12), followed by relapse for 2 pts (one concomitant with loss of BCA and one 7 months later). Three pts received a second infusion of CTL019 for loss of BCA with no further expansion of CAR-T cells. Prior treatment with blinatumomab was a significant predictive factor for relapse (HR=6.082, 95%CI, 1.2-30, p=0.0005) in a univariate analysis. There was a trend toward increased risk of relapse with increased disease burden (≥ 5%) before lymphodepletion regimen (HR=2.4, 95%CI, 0.7-8, p=0.14). Twenty-two pts experienced a CRS (≥ grade 3: n=13, all ≥ 10 y). ICU was required for 14 pts (34%). One 29 year-old pt died of an uncontrollable CRS at day 6. Ten pts received tocilizumab, 4 pts siltuximab and 9 pts corticosteroids. Age ≥ 10 y (p=0.04) and a high disease burden just before lymphodepletion (marrow blasts ≥ 5%) (p=0.01) were associated with a higher risk of CRS ≥ grade 3. Nine neurological events have been reported, being reversible except in 2 cases (one death in combination with grade 5 CRS-cf supra-, one HHV6-related encephalitis with neurological sequelae). Among the 9 pts who presented neurological events, 8 experienced CRS grade ≥ 3 (RR=17.2, 95%CI, 3.22-100.3, p=0.0001). By day 28, unresolved neutropenia grade ≥ 3 was reported for 13 pts. G-CSF treatment was required in 21 pts overall. Thrombocytopenia grade ≥3 was reported for 14 pts. Conclusion: CTL019 confirms its efficacy with a high response rate after infusion and very encouraging early outcomes in a cohort of pts heavily pretreated for refractory or multiply relapsed B-ALL. Persistent remissions with a potential for cure were observed without additional HSCT, relapses occurring within the first year after infusion of CTL019. Accurate assessment of a potentially deleterious effect of Blinatumomab preexposure notable on CD19 negative relapse will need more pts and a longer follow-up. Toxicity profile was tolerable and manageable thanks to collaboration between intensivists, neurologists and hematologists. The identification of severe CRS predictive risk factors (high disease burden just before lymphodepletion and age ≥ 10 y) points towards the reinforcement of toxicity monitoring and treatment anticipation in these cases. Disclosures Boissel: NOVARTIS: Consultancy. Baruchel:NOVARTIS: Consultancy.

Academic, Phase I/II Trial on T Cells Expressing a Second Generation, CD19-Specific Chimeric Antigen Receptor (CAR) and Inducible Caspase 9 Safety Switch for the Treatment of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) and B-Cell Non-Hodgkin Lymphoma (B-NHL) in Children

Survival rates of children with relapsed/refractory (r/r) BCP-ALL remain unsatisfactory and little progress has been made in the past 2 decades. Similarly, relapse of childhood B-NHL is usually associated with an aggressive disease and poor outcomes. Targeted immunotherapy with T-cells genetically modified to express a CD19-directed CAR showed an unprecedented antitumor efficacy, leading to the recent FDA and EMA approval of two CD19-CAR products for treatment of BCP-ALL and B-NHL. Relevant toxicities have, however, been reported, mainly related to the development of severe Cytokine Release Syndrome (CRS) and/or of neurotoxicity. At Ospedale Pediatrico Bambino Gesù (OPBG) in Rome, we developed a clinical-grade, 2nd generation, CD19-specific CAR construct, including 4.1bb as costimulatory domain and the inducible caspase-9 safety switch (iC9-CD19-CAR), vehiculated by a retroviral vector, to conduct an academic, phase I/II clinical trial in patients (age 1-25 yrs) affected by BCP-ALL or B-NHL. We now report on the results of the phase I and of the first 8 patients treated in the phase II portion of the study, in terms of feasibility, toxicity, maximum tolerated/recommended dose (MTD/RD) and data on response rate and biological correlates. The phase I, dose-escalation portion of the study included 3 dose levels (DL), namely: DL1, 0.5×106; DL2, 1.5×106; DL3, 3.0×106 CAR+ T cells per kg of recipient body weight. In the phase II portion, patients were treated at the RD identified in the phase I, namely 3.0×106 CAR+cells/kg. All patients received a lymphodepleting regimen consisting of fludarabine and cyclophosphamide for 3 days and iC9-CD19-CAR T cells were subsequently administered as single infusion. Patients were monitored for toxicity, expansion and persistence of iC9-CD19-CAR T cells. Seventeen children were enrolled into the trial and received iC9-CD19-CAR T cells between January 2018 and June 2019. Data were analyzed as of July 20, 2019. The characteristics of the patients are detailed in table 1. The designed dose concentration was successfully produced for all the enrolled patients and we did not observe any production failure. The median transduction rate in the drug product was 54% (range 21-73), while the median vector copy number was 3.8 (range 2.8-6.2). During the phase I portion of the study, no dose limiting toxicities (DLTs) have been recorded, defining the MTD as 3.0×106 CAR+ T cells per kg of recipient body weight. The treatment was overall tolerated and all the toxicities were reversible, the most severe being grade 3-4 neutropenia, thrombocytopenia and/or anemia, occurring in 16/17 (94.1%) patients; in 13/16 patients (81.2%) the hematological toxicity developed before the infusion and persisted after the administration of CAR T cells. Cytokine release syndrome (CRS) occurred in 10/17 patients (58.8%) and was overall moderate, reaching grade 3 (Lee criteria) in one patient only. Notably, none of the patients developed neurotoxicity and no activation of the safety switch was required. All patients were assessed for response at 4 weeks from iC9-CD19-CAR T cell infusion and 13/15 (86.7%) patients with ALL achieved complete remission (CR) with negativity of minimal residual disease (MRD), including 2/3 patients receiving the DL1, 9 patients who had failed a previous allogeneic haematopoietic stem-cell transplantation (HSCT) and 6 patients that had previously received blinatumomab, as CD19-directed immunotherapy. The iC9-CD19-CAR T cells expanded in vivo and were detectable by both flow-cytometry and molecular biology in the blood (Fig.1), bone marrow and cerebrospinal fluid of the responders. One CD19-negative relapse 3 months after infusion was recorded, while 3 additional patients relapsed with CD19+ leukemia blasts. Four patients received HSCT while in CR with MRD negativity because of regrowth of normal CD19+ B cells. The 18-month probability of overall survival for the BCP-ALL cohort is 72.2% (Fig.2). One of the 2 B-NHL patients showed a partial response. Our data indicate that iC9-CD19-CAR T cell in an academic setting is feasible, safe and extremely effective in treating highly resistant/relapsed BCP-ALL. In our trial, no major or life-threatening toxicities were observed and, despite the moderate CRS recorded, high rates of CR were achieved, suggesting that the combination of a retroviral platform and 4.1bb as costimulation is able to mediate a potent antitumor effect Disclosures Merli: Amgen: Honoraria; Novartis: Honoraria; Sobi: Consultancy; Bellicum: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Miltenyi: Honoraria. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

A Novel CD19-Anti-CD20 Bridging Protein Prevents and Reverses CD19-Negative Relapse from CAR19 T Cell Treatment In Vivo

Introduction CAR T cells that recognize the antigen CD19 (CAR19s) have achieved remarkable success in otherwise untreatable B cell malignancies including refractory and relapsed ALL and NHL. However, clinical data from diverse CAR19 trials, and real-world experience with the approved CAR19 therapeutics (tisagenlecleucel and axicabtagene ciloleucel), highlight a critical issue, that of patient relapse due to the loss of expression of the target antigen (CD19) or the antigenic epitope. Antigen loss relapse rate of up to 50% have been reported across indications (adult ALL, pediatric ALL, adult NHL) irrespective of the specific CAR19 used. Attempts to treat patients who have relapsed from CAR19 treatment include provision of a CAR T cell to a second antigen, for example CD22. Such attempts have met with limited success, further, many patients cannot tolerate a second regimen of apheresis, consolidation, lymphodepletion and CAR T infusion. Importantly, many of the patients relapsing with CD19-negative malignancies still have detectable levels of CAR19 T cells in circulation, since the CAR19s persist in the presence of normal B cells being produced by the bone marrow (these B cells are CD19-positive). Therefore, a technology that reactivates the patient-resident CAR19s to attack the relapsing tumor cell would be a highly attractive alternative to subsequent CAR T therapy. Here we present this technology and illustrate its' ability to prevent relapses and importantly, to reverse relapses in vivo. Experimental Procedures A stabilized form of the CD19 extracellular domain (ECD) was cloned in frame with an anti-CD20 scFv and an anti-albumin VHH, to create a monomeric CD19-ECD-anti-CD20 bridging protein with extended circulating half-life characteristics. The protein was purified from a mammalian cell expression system. Protein stability, binding affinities, and cytotoxic activity were analyzed in vitro. We utilized CD19-positive, CD20-positive and double positive cell lines to assess single and dual antigen activity. We utilized patient derived CD20-positive/CD19-negative cells to demonstrate translational relevance. Finally, we used single and dual flank in vivo models to assess the potency of the bridging protein in the relapse setting and in the prevention setting. Results and Discussion The CD19-anti-CD20 bridging protein was shown to be expressed at high levels, readily purified and highly stable (no aggregation or clipping, thermostable, and stable in media/serum at 37oC for extended periods). The purified bridging protein directed CAR19 cytotoxicity against CD19-negative/CD20-positive cells with superb potency (IC50 = 23pM = 1.6 ng/ml). CAR19 T cells that were previously activated by a CD19-positive tumor cell could subsequently be activated by a CD19-negative tumor cell in the presence of the CD19-anti-CD20 bridging protein. In vitro, a CAR19 T cells found and eliminated CD19-negative cells "hidden" in a population of dual-positive cells in a mixing experiment but only if the bridging protein was present, otherwise, the CD19-negative cells invariably escaped from CAR19 T cells. The activity of the CD19-anti-CD20 bridging protein extended to CD19-negative/CD20-positive patient-derived cells tested in vitro. In vivo, using a dual flank model, CAR19 T cells plus the injected bridging protein controlled both CD19-positive/CD20-positive and CD19-negative/CD20-positive tumors, while CAR19 alone did not impact the latter tumor. In a relapse setting the growth of a mixture of CD19-positive and CD19-negative cells was merely delayed by CAR19 T cells alone but was eradicated when CAR19 cells were given along with the CD19-anti-CD20 bridging protein injected systemically. Importantly, CAR19 cells that had "lost" control over the mixed population could be restimulated to eliminate the CD19-negative population when the CD19-anti-CD20 bridging protein was added after those cells have begun to escape the initial (CAR19-only) treatment in vivo. These results have led to the identification of a development candidate for the treatment of CD19-negative relapse from CAR19 treatment. The GMP production campaign is underway. The first-in-human trial will enroll patients relapsing from CAR19 therapy with CD19-negative malignancy, in whom CAR19 T cells are shown to still be present. Disclosures Rennert: Aleta Biotherapeutics: Employment, Equity Ownership. Su:Aleta Biotherapeutics: Employment. Dufort:Aleta Biotherapeutics: Employment. Birt:Aleta Biotherapeutics: Employment. Sanford:Aleta Biotherapeutics: Employment. Wu:Aleta Biotherapeutics: Employment. Ambrose:Aleta Biotherapeutics: Employment. Lobb:Aleta Biotherapeutics: Consultancy, Equity Ownership.

Cell Adhesion of ALL to Stromal Cells May Mediate CAR T-Cell Resistance: A Novel Escape Mechanism for Immunotherapy

Backgroud: Despite advances in therapy and improved survival, relapsed and refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL) in pediatric and adult patients still remains a problem. Chimeric antigen receptor T cells against CD19 (CD19 CAR T) show promising results in patients with r/r BCP-ALL. However, relapse of the disease still occurs with appreciable frequency even with this novel therapy. As a significant number of relapses post-CAR T lack surface CD19 expression, further CD19-directed therapy is not an option for these cases. Hypothesis: Sometimes despite CAR T engraftment and establishment of B-cell aplasia, relapse still occurs. We hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CAR T-cell resistance (CAM-CART-R) can contribute to relapse of ALL. Results: To test our hypothesis, primary ALL cells were treated with CD19 CAR T cells either with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures. We observed B-ALL cells treated with CD19 CAR T on OP9 has 10-20% higher viability compared to B-ALL and CD19 CAR T co-culture in medium alone, supporting the notion of CAM-CART-R. We also determined that soluble factors in OP9 primed medium may contribute to CAM-CART-R. However, the direct stromal contact mediated significant protection again CAR T induced apoptosis of B-ALL cells. To determine the molecular mechanisms underlying the survival promoting effects of stromal cells on CD19-, these cells were starved in serum-free media for 4hours and then treated with PI3Kδ inhibitor CAL-101 or DMSO and co-cultured with OP9 cells for 1 hour. We found that p-Akt is upregulated by stromal contact in CD19-negative B-ALL cells post-CAR T therapy and that PI3Kδ inhibition using can downregulate p-Akt in CD19-negative B-ALL patients. Critically, we investigated whether CD19 CAR T cells were functional under these conditions. For this purpose, we determined if stromal contact of ALL cells or stromal contact of CAR T cells changes the intracellular cytokine milieu of CD19 CAR T cells and found that intracellular IL-6, TNF- α and IFN-γ were reduced upon stromal contact supporting our hypothesis of a role of stromal cells in CAM-CART-R. We also determined that immune checkpoints molecules on T cells are unaffected by OP9 cells. Despite the reduction of cytokine level in T cells upon co-culture with B-ALL cells on OP9, PD-1, TIM-3 and LAG3 expression on CD19 CAR T cells after 2 days of co-culture was not altered as determined by flow cytometry. Resistance of ALL cells to CD19 CART cells was not mediated through checkpoint inhibition, since the PD-1/PD-L1 inhibitor Nivolumab failed to enhance ALL killing. Phenotypic profiling of thirteen cases of primary ALL relapse post-CD19 CAR T cell therapy showed high expression of adhesion molecules including integrin α4. Phenotypic analysis also revealed high expression of integrins is retained in primary ALL cells after CD19 knockout in one case. To explore possible solutions to overcome CAM-CART-R, we examined a strategy of blocking specifically integrin α4. We have previously shown that blocking integrin α4 can de-adhere CD19-negative B-ALL relapse post-CAR T cell therapy from their respective counter-ligands in vitro and that these cells can benefit from integrin blocking therapy in vivo. We have now confirmed this in NSG mice injected with CD19-negative B-ALL cells from a patient with post-CAR T cell relapse. Mice were treated intraperitoneally (n=6/group) with total immunoglobulin (Ig) control or humanized anti-human integrin α4 antibody Natalizumab (NZM). As a result, Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control Ig group (66 days (Ig) vs 85 days (NZM) p&lt;0.005). Further combination treatments with chemotherapy are in progress. Conclusion: In summary, our data indicate that similarly to CAM-DR, CAM-CART-R can occur resulting in relapse of ALL. Targeting adhesion molecules may be a new approach to treat or prevent relapse following CD19 CAR T cell therapy for . Disclosures Ahmed: CellMedica: Other: Royalties; Celgene: Other: Royalties; Adaptimmune: Membership on an entity's Board of Directors or advisory committees. Babak:Simurx. Inc: Other: Founder . Pulsipher: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; Amgen: Other: Lecture; Bellicum: Consultancy; Miltenyi: Research Funding; Medac: Honoraria. Wayne:AbbVie: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Servier: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding. Abdel-Azim:Adaptive: Research Funding.

Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with &amp;gt;5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

Clinical Experience of CAR T Cell Immunotherapy for Relapsed and Refractory Infant ALL Demonstrates Feasibility and Favorable Responses

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.

AUTO1, a Novel Fast Off CD19CAR Delivers Durable Remissions and Prolonged CAR T Cell Persistence with Low CRS or Neurotoxicity in Adult ALL

Introduction: In adults, prognosis for B-ALL is poor, patients are more vulnerable to CD19 CAR immunotoxicity and there is currently no CD19 CAR therapeutic with acceptable toxicity and durable efficacy. We have developed a novel second generation CD19CAR (CAT-41BBz CAR), with a faster off-rate but equivalent on rate than the FMC63-41BBz CAR (Kd 116 nM vs 0.9 nM, T1/2 9s vs 4.2 hours) designed to result in more physiological T-cell activation, reduce toxicity and improve engraftment. Preliminary paediatric clinical data of this novel CD19 CAR (AUTO1) supports this assertion. We here describe preliminary data from ALLCAR19 (NCT02935257), a multi-centre, Phase I clinical study of AUTO1 as therapy for r/r adult B-ALL. Methods: Manufacturing: AUTO1 utilises non-mobilised autologous leucapheresate. The first 6 trial products were generated using a standard dynal bead/WAVE Bioreactor process and subsequent products using a semi-automated closed process. Study design: ALLCAR19 is a phase I/II study recruiting subjects 16-65y with r/r B ALL. Lymphodepletion with fludarabine (30mg/m2 x3) and cyclophosphamide (60mg/kg x1) is followed by split dose CAR T cell infusion (Day 0: if ≥20% BM blasts, infuse 10 x 106 CAR T cells ; if &amp;lt;20% BM blasts, infuse 100 x 106 CAR T cells. Day +9: if no Grade 3-5 CRS/CRES, infuse Dose 2, to a total dose of 410 x 106 CAR T cells). Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months Results: As of 24 July 2019, 16 patients have been leukaphresed, 14 products manufactured (one failed leukaphresis and one currently in manufacture) and 13 patients have received at least 1 dose of AUTO1. Of the 16 patients, median age was 35.5 (range 18-63), 10/16 (63%) had prior blinatumomab or inotuzumab ozogamicin and 12/16 (75%) had prior HSCT. At the time of pre-conditioning, 9/13 (69%) patients were in morphological relapse with &amp;gt;5% leukemic blasts of which 6/13 (46%) had ≥50% blast. 9/13 patients (69%) received the total target split dose of 410 x 106 CAR T cells while 1/13 patients (8%) received a reduced split total dose of 51.3 x 106 CAR T cells due to manufacturing constraints. 3/13 patients (23%) received only a first dose of 10 x 106 CAR T cells. The dose was administered safely to date: No patients experienced ≥Grade 3 CRS (using Lee criteria) and only 1/13 (8%) experienced Grade 3 neurotoxicity (dysphasia) that resolved swiftly with steroids. All patients had robust CAR expansion (median peak expansion 172 CAR/uL blood). Of the 13 patients dosed (1/13 pending 28 day follow up), 10/12 (83%) achieved MRD negative CR at 1 month and all patients had ongoing CAR T cell persistence at last follow up. Two patients experienced CD19 negative relapse (one at M3, one at M6), 1 patient died on D17 before first response evaluation, 1 died in molecular CR from sepsis, and 1 died from persistent disease. Currently, 7/12 remain on study and continue in flow/molecular MRD negative remission with a median follow up of 9.0 months (range 1.2-14.8). Conclusions: AUTO1 delivers excellent early remission rates with initial data showing 83% MRD negative CR and robust CAR expansion and persistence. Despite high tumour burden, the safety profile compares favourably to other CD19 CARs, with no cases of severe CRS and only one case of Gr3 neurotoxicity. This is consistent with experience in the paediatric cohort. Updated results will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

Allogeneic CD34-Selected HSCT Following CAR T-Cells Is Associated with Low TRM and Favorable OS in Pediatric/Young Adult Patients with Relapsed/Refractory B-ALL

Background: Chimeric antigen receptor (CAR) T-cells have transformed the therapeutic options for patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). The tolerability and outcome of allo-HSCT following CAR T-cell therapy is yet to be defined. Methods: We retrospectively reviewed pediatric and young adult patients with R/R B-ALL who received allo-HSCT following CAR T-cells. Outcomes of interest were overall survival (OS), cumulative incidence of relapse, and transplant-related mortality (TRM). These were estimated using log-rank analysis. Variables of interest were disease status at time of allo-HSCT, time from CAR T-cell therapy to start of allo-HSCT, conditioning regimen, graft source and manipulation, GVHD prophylaxis, donor HLA matching, neutrophil/platelet engraftment, relapse and cause of death. Results: Sixteen patients, with a median age of 13 (range 1-20), at time of allo-HSCT were included. All patients achieved MRD-negative complete remission (CR) following CAR T-cells and proceeded to allo-HSCT as definitive therapy. CR status included CR1 (n=6), CR2 (n=5), CR3 (n=4), and CR4 (n=1). The median time from CAR T-cells to the start of allo-HSCT conditioning was 46.5 days (range 20 -126). Conditioning was TBI-based (n=13) or chemotherapy only (n=3). Graft source included PBSC (n=8) or BM (n=8). Graft manipulation included CD34-selected T-cell depleted (TCD) (Miltenyi) (n=9) or an un-modified HSCT (n=7). Donors were HLA-matched related (n=8), HLA-matched unrelated (n=3), or HLA-mismatched unrelated (n=5). GVHD prophylaxis in the non-manipulated grafts included a calcineurin inhibitor (CNI) plus MTX (n=5), CNI plus MMF (n=1) or post-transplant cyclophosphamide (n=1). Available data included: 15/15 patients achieved neutrophil engraftment (median 12 days; range 9-29) and 13/15 patients achieved platelet engraftment (median 30 days; range 16-265). The 12-month cumulative incidence of relapse was 0% in patients receiving CD34-selected transplant, versus 25% [95% CI: 0 to 67%] in patients receiving un-modified transplant (log-rank p=0.80) and the 24-month cumulative incidence of relapse for the whole cohort was 25% [0-47%]. Late relapse (&gt;12 months from allo-HSCT) occurred in three (n=3) patients in the CD34-selected TCD cohort with a median time to relapse from allo-HSCT of 24.2 months (range 16.3 - 24.7 months). Two (n=2) patients achieved subsequent MRD-neg/CR following additional CD19-specific CAR T-cell infusions (CRs ongoing at 12.4 and 1.4 months). The other patient (n=1) experienced a CD19-negative relapse and is awaiting infusion of CD22-specific CAR T-cells. Early relapse occurred in one (n=1) patient (4.7 months from allo-HSCT) following un-modified allo-HSCT with subsequent MRD-neg/CR following treatment with CD19-specific CAR T-cells (CR ongoing at 5.2 month). Three (n=3) patients in the un-modified cohort died due to TRM (24-month TRM = 46% [7-86%]), including veno-occlusive disease (multi-organ failure/infection) (n=2) and GVHD (n=1), while no TRM was noted in the CD34-selected TCD cohort (p=0.03). The TRM for the entire cohort was 19% at 24 months [0-39%]. The 24-month OS for the entire cohort was 81% [61 -100%], with 100% OS in CD34-selected TCD recipients and 54% in the un-modified recipients (p=0.03). Conclusion: CAR T-cells when followed by a CD34-selected TCD allo-HSCT resulted in less TRM and favorable OS when compared to un-modified allo-HSCT. Disease control is not impacted by the type of allo-HSCT and late relapse was seen following CD34-selected TCD. Successful re-treatment with CAR T-cells to induce remission is possible and analysis as to the durability of this response is ongoing. Disclosures Margossian: Novartis: Membership on an entity's Board of Directors or advisory committees. Prockop:Atara Biotherapeutics: Other: Support for industry sponsored trails ; Mesoblast: Other: Support for industry sponsored trails . Shah:Jazz pharmaceuticals: Speakers Bureau. Boelens:Takeda: Consultancy; Magenta: Consultancy; Bluerock: Consultancy; Avrobio: Consultancy. Curran:Juno Therapeutics: Consultancy, Research Funding; Novartis: Consultancy.

A Phase 2, Open-Label, Multicenter Study Evaluating the Safety and Efficacy of Axicabtagene Ciloleucel in Combination with Either Rituximab or Lenalidomide in Patients with Refractory Large B-Cell Lymphoma (ZUMA-14)

A Phase 2, Open-Label, Multicenter Study Evaluating the Safety and Efficacy of Axicabtagene Ciloleucel in Combination with Either Rituximab or Lenalidomide in Patients with Refractory Large B-Cell Lymphoma (ZUMA-14)

Bispecific Chimeric Antigen Receptor Targeting Both CD19 and CD22 T Cell Therapy in Adults with Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

Background: Despite the impressive CR induced by CD19 CAR-T cell therapy in B-ALL, the high rate of complete responses is sometimes limited by the emergence of CD19-negative leukemia. Bispecific CAR-modified T cells targeting both CD19 and CD22 may overcome the limitation of CD19-negative relapse. Methods: Here, we report the design of a bispecific CAR that triggers robust cytolytic activity against target cells by targeting either CD19 or CD22. We performed a phase 1 trial of bispecific CAR T cell therapy in patients with relapsed/refractory precursor B-ALL at a dose that ranged from 1.7×106 to 3×106 CAR T cells per kilogram of body weight. Finding: MRD-negative CR was achieved in 6 out of 6 enrolled patients. Autologous CD19/CD22 CAR T cells proliferated in vivo and were detected in the blood, bone marrow, and cerebrospinal fluid. No neurotoxicity occurred in any of the 6 patients treated. Of note, one patient had a relapse with blast cells that no longer expressed CD19 and exhibited diminished CD22 site density approximately 5 months after treatment. Interpretation: In brief, autologous CD19/CD22 CAR T cell therapy is feasible and safe and mediates potent antileukemic activity in patients with relapsed/refractory B-ALL. Furthermore, the emergence of target antigen loss and expression downregulation highlights the critical need to anticipate antigen escape. Our study demonstrates the reliability of bispecific CD19/CD22 CAR T cell therapy in inducing remission in adult patients with relapsed/refractory B-ALL. Trial Registration: NCT03185494. Funding Statement: National Key Research and Development Program of China and National Natural Science Foundation of China. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocols were approved by the institutional review board at the Chinese PLA general hospital, and the patients provided written informed consent. This clinical investigation was conducted according to the principles of the Declaration of Helsinki.