Abstract: The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

Abstract: Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has significantly improved outcomes in the treatment of refractory or relapsed large B-cell lymphoma (LBCL). We evaluated the long-term course of hematologic recovery, immune reconstitution, and infectious complications in 41 patients with LBCL treated with axicabtagene ciloleucel (axi-cel) at a single center. Grade 3+ cytopenias occurred in 97.6% of patients within the first 28 days postinfusion, with most resolved by 6 months. Overall, 63.4% of patients received a red blood cell transfusion, 34.1% of patients received a platelet transfusion, 36.6% of patients received IV immunoglobulin, and 51.2% of patients received growth factor (granulocyte colony-stimulating factor) injections beyond the first 28 days postinfusion. Only 40% of patients had recovered detectable CD19+ B cells by 1 year, and 50% of patients had a CD4+ T-cell count & lt;200 cells per μL by 18 months postinfusion. Patients with durable responses to axi-cel had significantly longer durations of B-cell aplasia, and this duration correlated strongly with the recovery of CD4+ T-cell counts. There were significantly more infections within the first 28 days compared with any other period of follow-up, with the majority being mild-moderate in severity. Receipt of corticosteroids was the only factor that predicted risk of infection in a multivariate analysis (hazard ratio, 3.69; 95% confidence interval, 1.18-16.5). Opportunistic infections due to Pneumocystis jirovecii and varicella-zoster virus occurred up to 18 months postinfusion in patients who prematurely discontinued prophylaxis. These results support the use of comprehensive supportive care, including long-term monitoring and antimicrobial prophylaxis, beyond 12 months after axi-cel treatment.

Abstract: Bleeding and thrombotic events are an emerging toxicity associated with chimeric antigen receptor (CAR) therapies. To determine their incidence, we retrospectively analyzed consecutive adult patients (N = 127) with large B-cell lymphoma (LBCL) or B-cell acute lymphoblastic leukemia (B-ALL) treated from 2017 through 2020 with axicabtagene ciloleucel (axi-cel; n = 89) or a bispecific CD19/CD22 CAR (n = 38). Twelve (9.4%) and 8 (6.3%) patients developed bleeding and thrombosis within the first 3 months, respectively. In the axi-cel subgroup, these occurred in 11.2% and 6.7%, respectively. Bleeding occurred between days 8 and 30 (median, 17.5) and thrombosis between days 2 and 91 (median, 29). Bleeding sites included genitourinary, soft tissue, intracranial, gastrointestinal, and pulmonary and were associated with features of consumptive coagulopathy. On univariate analysis, patients with bleeding were older, had lower baseline platelets (86 × 103/μL vs 178 × 103/μL; P & lt; .01), lower platelet and fibrinogen nadirs , and elevated lactate dehydrogenase. Immune effector cell (IEC)–associated neurotoxicity syndrome (ICANS) grade ≥3 was associated with increased bleeding (50% vs 15%; P = .01), thrombosis (50% vs 16%; P = .04), prothrombin time prolongation, hypofibrinogenemia, and elevated D-dimer. Low pretreatment platelet counts were associated with bleeding in a multivariate logistic regression model. Patients with thrombocytopenia or severe ICANS are at increased risk of bleeding and should be closely monitored, particularly within the first month after CAR therapy. Future studies in larger cohorts should assess risk factors for systemic coagulopathies in CAR T therapy, including their association with neurotoxicity.

Abstract: Background: Axicabtagene ciloleucel (axi-cel), an autologous anti-CD19 chimeric antigen receptor (CAR-T), showed significant clinical responses in patients with relapsed-refractory large-B cell lymphomas in the Zuma-1 trial (Neelapu et al, NEJM 2017). Zuma-1 analysis showed blood CAR-T cell expansion was associated with clinical response and toxicity. Herein, we report on 25 patients treated with commercial axi-cel and describe CAR-T expansion by immunophenotyping and its correlation with clinical outcomes. Methods: Twenty-five patients with aggressive lymphoma consecutively apheresed at Stanford University prior to June 30, 2018 were studied on an IRB approved biorepository-clinical outcome protocol. Cytokine release syndrome (CRS) was graded by Lee criteria (Blood 2014) and neurotoxicity according to Neelapu et. al (Nat. Rev. Clin. Onc. 2017). CAR-T cell immunophenotyping was assessed by peripheral blood flow cytometry on days 7, 14, 21 and 28 and then monthly. CAR-T cells were identified by gating on singlet+, live+, CD45+, CD14-, CD3+, anti-CD19-specific CAR mAb (clone 136.20.1; Jena et. al Plos 2013) and characterized as either CD4+ or CD8+. Results: Of 25 apheresed patients, 3 patients died prior to axi-cel infusion due to progressive lymphoma. Of 22 infused patients, 14 (64%) would have been eligible for the Zuma-1 trial. Reasons for ineligibility included symptomatic DVT (n=2), renal insufficiency (n=1), transaminitis (n=1), thrombocytopenia (n=1), MDS (n=1), pleural effusion (n=1) and 1 was ineligible by multiple criteria. Median time from initial clinic visit to infusion was 47 days (range 34-117); median time from apheresis to infusion was 22 days (range 19-38). Nine patients received bridging therapy prior to lymphodepletion chemotherapy (chemo = 4, radiation = 2, high dose dexamethasone = 3). Axi-cel infusion occurred in hospital and patients were followed expectantly for a minimum of 7 days or until adverse events resolved to 〈 Grade 2; median hospitalization was 13.5 days (range 7-44). Ninety-five percent of patients developed CRS (Grade 2 = 73%, none ≥Grade 3). Median number of tocilizumab doses was 1 (range 0-4). Neurotoxicity occurred in 64%, Grade 3 or 4 in 27%. Corticosteroid therapy was required in 82% (77% received both tocilizumab and steroids). Median duration of steroids was 8.5 days (range 1-30); 12 patients required at least 1 week and 4 patients ≥2 weeks. Of patients infused, complete response (CR) at day 28 was 45% (ORR 86%). Of 15 patients evaluable at 3 months, ORR was 53% (CR = 7, PR = 1) and 47% progressed, similar to Zuma-1. Ineligibility for Zuma-1 was not associated with inferior outcomes. Overall, median day 7 peak in vivo axi-cel expansion using anti-CAR19 flow cytometry was 38 CAR-T/ul (Fig. 1A), matching RT-PCR measured levels reported in Zuma-1. As shown in Fig. 1A, the majority of CAR-T cells were CD8+. Patients with Grade 2 CRS had significantly higher peak expansion of CAR-T cells (both CD4+ and CD8+) as compared to those with either Grade 0 or 1 CRS (Fig. 1B). Grades 2-4 neurotoxicity were significantly associated with peak total and CD8+ CAR-T but not CD4+ (Fig. 1B). Illustratively, 2 patients with the most robust CAR-T expansion (▪, ▼ Fig. 1A) experienced Grade 4 neurotoxicity including status epilepticus requiring multiple anti-epileptics and intubation. Peak CAR-T expansion in blood did not correlate with CR or ORR at day 28; expansion did not differ between patients who did or did not require steroids. Fine needle aspirates (FNA) on a subset of patients with FDG-avid lymph nodes 2-3 days post axi-cel showed significant CAR-T expansion within the node despite low detectable circulating CAR-T. Figure 1C depicts a 76-year-old male with double expressor DLBCL who attained a CR at day 28; day 14 blood CAR-T expansion was below average (6 CAR-T/ul), while his day 2 FNA showed 〉 35% of CD3+ T-cells expressed CAR19. As of submission, 34 patients were apheresed and updated blood and FNA results will be presented. Conclusion: Our analysis of 22 infused axi-cel patients showed an ORR of 86% and CR of 45%, despite 36% Zuma-1 ineligibilities and steroid use in 82%. Blood CAR-T expansion was associated with both CRS and neurotoxicity but not clinical response. Detection of high concentration of CAR-T cells in affected lymph nodes 2 days post infusion suggests quantification of CAR-T cells at disease sites could be predictive of clinical responses. J.Y.S and B.S are co-first authors Disclosures Latchford: Kite a Gilead Company: Speakers Bureau. Muffly:Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding. Miklos:Kite - Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Research Funding; Novartis: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding.

Abstract: Background: The prognostic and predictive utility of measurable residual disease (MRD) assessments using bone marrow (BM) aspirates is well-established in the management of acute lymphoblastic leukemia (ALL). However, frequent BM MRD monitoring post-therapy is limited by the invasive, expensive, and at times impractical nature of numerous BM examinations. Limited retrospective reports have suggested that MRD analysis by next-generation sequencing (NGS) using peripheral blood (PB) may provide a viable alternative to MRD monitoring of the BM. We conducted a prospective, multi-institutional observational study of NGS-based MRD of the PB among adult ALL patients undergoing cellular therapies (hematopoietic cell transplantation [HCT] and chimeric antigen receptor T cells [CART] ) in order to determine the correlation between PB and BM MRD and to explore the clinical utility of monitoring MRD in the PB. Methods: Patients & gt;= 18 years-old with ALL were recruited from Stanford University and Kaiser Permanente, Northern California. The MRD analyses were conducted using Adaptive Biotechnologies ClonoSEQ NGS based platform that tracks tumor specific VDJ rearrangement in B and T cell malignancies. Assessment of MRD was obtained from the PB and BM prior to HCT/CART. Among HCT patients, PB MRD was obtained at one month, and then every 2-3 months for the first year following HCT; a paired BM MRD sample was obtained at the 3 month time-point with optional additional BM examinations. Among CART patients, paired PB and BM MRD were obtained at one month, and then every 2-3 months for the first year following CART. The correlation between log10 values of PB and BM MRD samples was evaluated using the Pearson correlation coefficient. Clinical relapse was defined as morphologic leukemia blasts in the marrow or extramedullary site, or administration of a new therapy for rising MRD. Results: Sixty-nine patients scheduled to undergo cellular therapies were enrolled; 3 (4%) did not undergo planned therapy and were excluded and 4 (6%) lacked a detectable clonal leukemia sequence and were thus off study, resulting in a study population of 62 (42 BMT, 17 CART, 3 BMT and CART). The median age was 42 years (IQR 30-53), 36 (58%) were male, 54 (87%) had B-ALL,16 (26%) were BCR-ABL+, and 28 (46%) had extramedullary (EM) involvement. Across all patients, PB MRD was highly correlated with BM MRD (r=0.87; p & lt;0.0001; Figure 1A). Of the 129 paired samples,15 (12%) had discordance with MRD identified in either the PB and not BM (N=7; 5%) or in the BM and not PB (N=8; 6%). Similarly, PB and BM MRD were highly correlated in the HCT (r=0.86; p & lt;0.0001) and CART cohorts (r=0.86; p & lt;0.001; Figure 1B), and among patients with EM involvement (r=0.83; p & lt;0.0001) and marrow only disease (r=0.93; p & lt;0.0001; Figure 1C). With median follow-up of 293 days (IQR: 180-512), 6 (13%) HCT and 13 (65%) CART patients experienced clinical relapse (Figure 2A). Among the 6 patients who relapsed following HCT, 80% had detectable MRD in the PB prior to HCT (1 was missing pre-HCT PB sample). Following HCT, all 6 patients developed detectable MRD, with median time from first MRD positivity to clinical relapse of 71 days (IQR 28-90). Among the 13 patients who relapsed following CART, 85% had detectable MRD in the PB a median of 60 days (50-139) prior to clinical relapse. Finally, serial monitoring of the PB following HCT averted clinical relapse through immunosuppression withdrawal in two patients with rising MRD post-HCT (Figure 2B), thereby directly impacting patient outcomes. Conclusion: This prospective observational study demonstrates a strong correlation between PB and BM NGS MRD results in ALL. These results show that non-invasive monitoring of PB-based MRD in ALL patients undergoing curative intent cellular therapies represents a viable alternative to serial BM examinations, providing clinically actionable information and the opportunity to intervene on impending clinical relapse. Disclosures Muffly: Adaptive: Research Funding; Amgen: Consultancy; Servier: Research Funding. Meyer:Orca Bio: Research Funding. Negrin:Amgen: Consultancy; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company; Biosource: Current equity holder in private company; UpToDate: Honoraria; KUUR Therapeutics: Consultancy. Rezvani:Pharmacyclics: Research Funding. Sidana:Janssen: Consultancy. Shiraz:ORCA BioSystems: Research Funding; Kite, a Gilead Company: Research Funding. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Liedtke:Adaptive: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria. Miklos:Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding.

Abstract: Background Treatment with chimeric antigen receptor (CAR) T cell therapies have shown dramatic, often durable responses for relapsed/refractory B-cell malignancies. However, it can be associated with significant side effects such as cytokine release syndrome (CRS), immune effector-cell associated neurotoxicity syndrome (ICANS) and life-threatening consumptive coagulopathies. The underlying pathobiology of such hemostatic defects and their distinct clinical sequelae remains obscure. This retrospective study aims at quantifying CAR T therapy associated bleeding and thrombotic complications and their association with CRS, ICANS, and laboratory derangements. Methods 130 adult patients with DLBCL or B-ALL treated between 2017-2020 with CD19 CAR-T therapy axicabtagene ciloleucel (N=90) or a bispecific CD 19/22 CAR construct utilizing 4-1BB costimulatory domains (N=40) were analyzed to determine dynamics of coagulation parameters and platelet counts as well as incidences of bleeding or thrombosis in the first three months after CAR T infusion. Events were included if graded ≥ 2 or if intervention was required. Platelet counts and coagulation parameters were collected prior to lymphodepletion (pre-LD), day 0, 3, 7, 14, 21, 28, 60 and 90. Results 12 (9.2%) and 8 (6.2%) patients developed bleeding and thrombotic complications in the first three months after CAR-T infusion, respectively. Events are characterized in Figure 1. All bleeding events occurred between days 0-30 (median 17.5, range 8-30), while thrombotic events occurred between days 2-91 (median day 29, range, 2-91). Two (1.5%) patients experienced both bleeding and thrombosis. Bleeding events coincided with the onset of thrombocytopenia and hypofibrinogenemia, and patients who bled had lower platelet (median 22.5 vs. 47 K/uL; p=0.03) and fibrinogen (median 151 vs. 351 ug/mL; p=0.007) nadirs in the first 30 days compared to those without bleeding. Temporally, the lowest median platelet nadir occurred at day 7 in patients with bleeding events vs. day 21 in patients without bleeding, while timing of fibrinogen nadirs were at day 21 in both. Patients with bleeding episodes were more likely to be older (median age: 70 vs. 60 yrs, p=0.03), have thrombocytopenia prior to lymphodepletion therapy (median 117.5 vs. 174.5 K/uL, p=0.01), and have elevated LDH (lymphoma subgroup; p=0.07). Other lab derangements in the first 30 days seen more frequently in patients with bleeding included prolonged thrombin time (TT) (21% vs. 6%; p=0.02), PT (16% vs. 5%; p=0.06), and elevated d-dimer (16% vs. 3%; p=0.01) indicative of a consumptive process. Thrombotic events were not significantly associated with elevated or peak d-dimer values (median 4.97 vs. 2.37 ug/mL, p=0.20). Interestingly, occurrence or severity of CRS was not associated with bleeding or thrombotic events, nor was it associated with marked derangements in coagulation abnormalities. However, higher grade ICANS (grade & gt; 3) was associated with bleeding (42% vs. 15%; p=0.038), thrombosis (50% vs. 16%; p=0.03), and evidence of endothelial activation including PT prolongation (78% vs. 35%; p & lt;0.001), hypofibrinogenemia (57% vs. 20%; p=0.001), and trend towards elevated d-dimer (70% vs. 46%; p=0.06). 13 (10%) patients received anticoagulation for prophylaxis or therapeutic indications that predated CAR T infusion. Four started anticoagulation secondarily for thrombotic events after CAR-T infusion, and one received tissue plasminogen activator (tPA) for an acute stroke. In this group, no patients developed bleeding complications from anticoagulation. Conclusion Both bleeding (9.2%), and thrombotic (6.2%) events are observed after CAR T cell therapy, with bleeding limited to the first month in our cohort. Notably, ICANS was uniquely associated with PT prolongation, hypofibrinogenemia, and increased fibrin degradation, in addition to both bleeding and thrombosis. These results suggest that a systemic coagulopathy coincides with high grade ICANS and whether these neurologic events truly represent sequelae of widespread vascular dysfunction warrants further investigation. Anticoagulation was safe in the patients whom it was indicated. Risk factors for bleeding and thrombotic complications should be studied prospectively to develop risk-assessment models and clinical guidelines for management of bleeding and thrombosis (including prophylaxis) during CAR T therapy. Disclosures Muffly: Adaptive: Research Funding; Servier: Research Funding; Amgen: Consultancy. Negrin:BioEclipse Therapeutics: Current equity holder in private company; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; KUUR Therapeutics: Consultancy; Biosource: Current equity holder in private company; Amgen: Consultancy; UpToDate: Honoraria. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Meyer:Orca Bio: Research Funding. Shiraz:Kite, a Gilead Company: Research Funding; ORCA BioSystems: Research Funding. Rezvani:Pharmacyclics: Research Funding. Mackall:Apricity Health: Consultancy, Current equity holder in private company; NeoImmune Tech: Consultancy; Nektar Therapeutics: Consultancy; Allogene: Current equity holder in publicly-traded company; BMS: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company. Miklos:Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding. Sidana:Janssen: Consultancy.

Abstract: BACKGROUND: Patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) after failure of CD19-directed CAR T-cell therapy (CAR19) have a dire prognosis, with an overall response rate (ORR) of 29% to conventional salvage therapies, and a median overall survival (OS) of 6 months. CD22 is expressed on the majority of B-cell malignancies. Autologous CAR T-cells targeting CD22 (CAR22) have yielded an ORR of 70-90% in pediatric patients with R/R B-cell acute lymphoblastic leukemia (B-ALL), including those who had previously failed CAR19 therapy. Based on these encouraging results, we evaluated CAR22 in adult patients with R/R LBCL, focusing on those with CAR19-refractory disease. METHODS: This ongoing single-institution phase I dose escalation clinical trial (NCT04088890) is evaluating a CAR construct incorporating the m971 CD22 single chain variable fragments and 41BB/CD3z endodomains integrated within autologous T-cells via lentiviral transduction. After lymphodepletion (LD) with fludarabine and cyclophosphamide, patients are infused with cryopreserved CAR T-cells after a 7- to 11-day closed manufacturing process utilizing the CliniMACS Prodigy device (Miltenyi). Primary objectives assess the ability to successfully manufacture CAR22 and safety. Secondary objectives include efficacy and durability of responses. RESULTS: Twenty-one patients with LBCL [n=12 at dose level 1 (DL1), 1x10 6 CAR+ cells/kg; n=9 at dose level 2 (DL2), 3x10 6 CAR+ cells/kg] have been enrolled with a median age of 64 years (range, 36-79) and a median of 4 (range, 3-8) prior lines of therapy. All patients had at least one high risk feature, including failure of prior CAR19 therapy (n=20); refractory disease to second-line or later therapy (n=17); elevated lactate dehydrogenase (LDH) pre-LD (n=17); high tumor burden (n=9); a history of primary refractory disease (n=7); failure of prior autologous hematopoietic stem cell transplantation (HSCT) (n=6); never achieving CR to any therapy (n=5); or LBCL with MYC gene rearrangements (n=5). Successful manufacturing of cells was achieved in all patients. All patients reached day 28 post-infusion and are included in the safety and efficacy analysis presented here; updated results will be presented at the meeting. Every patient experienced cytokine release syndrome (CRS); 20/21 (95%) were Grade 1-2, 1/21 (5%) were Grade 3. Four patients (19%) experienced immune effector cell-associated neurotoxicity syndrome (ICANS); all cases were Grade 1-2 and resolved within 2 days. Five patients (24%) experienced a hyperinflammatory macrophage activation syndrome (MAS), manifested in all cases by pancytopenia and consumptive coagulopathy (DIC) requiring transfusion and/or growth factor support. One patient who received DL2 had a Grade 5 infectious event in the setting of ongoing MAS and pancytopenia. Relative to DL1, higher prevalence of Grade ≥3 cytopenias beyond D28 (89% vs. 50%) and MAS (33% vs. 17%) were observed at DL2; thus, DL1 was selected as the maximally tolerated dose (MTD). ORR at D28 was 86% (CR, n=11; PR, n=7), and was similar between DL1 and DL2 (92% vs. 78%; p=ns). 3/7 (43%) initial PR improved to CR at a median of 3 months post-infusion. All 14 patients (67% of cohort) who achieved CR remain in remission, with a mean follow-up of 7.3 months (range, 1.2-21.3); median progression free survival (PFS) and OS have not yet been reached. Five patients died from disease progression, and one patient died from septic shock in CR. CD22 expression by flow was downregulated or absent in 1/3 (33%) patients evaluated after relapse. Peak CAR-T expansion as detected by peripheral blood flow cytometry occurred at a median of 14 days, with a trend towards earlier and higher peak levels in DL2 patients. Significantly higher mean CAR-T levels occurred at peak expansion in patients who developed MAS (1070±915 vs. 196±209 CAR+ cells/μL; p=0.001). CONCLUSIONS: Infusion of CAR22 in R/R LBCL is safe and well tolerated at DL1. Manufacturing of CAR22 was uniformly successful. With a mean follow-up of 7.3 months, the ORR and CR rates are 18/21 (86%) and 14/21 (67%), respectively. These data demonstrate CAR22 to be an effective salvage therapy for CAR19-refractory or CD19-negative LBCL. Figure 1 Figure 1. Disclosures Frank: Allogene Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees. Oak: Kite Pharma-Gilead: Research Funding. Arai: Magenta Therapeutics: Research Funding. Rezvani: Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding; US Department of Justice: Consultancy. Shiraz: Kite Pharma-Gilead: Research Funding. Sidana: Janssen: Consultancy, Research Funding; Allogene: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; BMS: Consultancy. Weng: Kite Pharma: Research Funding. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding. Feldman: Samsara Biocapital: Consultancy; Obsidian: Consultancy; Lonza PerMed: Consultancy; Gradalis: Consultancy. Mackall: Lyell: Consultancy, Current equity holder in publicly-traded company, Other: Founder; Syncopation Life Sciences: Consultancy, Current holder of individual stocks in a privately-held company, Other: Founder; Apricity: Consultancy, Current equity holder in publicly-traded company; Neoimmune Tech: Consultancy; Nektar: Consultancy, Research Funding. Miklos: Kite, a Gilead Company, Amgen, Atara, Wugen, Celgene, Novartis, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Bioscience, Adicet, Pharmacyclics, Janssen, Takeda, Adaptive Biotechnologies and Miltenyi Biotechnologies: Consultancy; Pharmacyclics: Patents & Royalties; Pharmacyclics, Amgen, Kite, a Gilead Company, Novartis, Roche, Genentech, Becton Dickinson, Isoplexis, Miltenyi, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Biosciences, Adicet, Adaptive Biotechnologies: Research Funding; Adaptive Biotechnologies, Novartis, Juno/Celgene-BMS, Kite, a Gilead Company, Pharmacyclics-AbbVie, Janssen, Pharmacyclics, AlloGene, Precision Bioscience, Miltenyi Biotech, Adicet, Takeda: Membership on an entity's Board of Directors or advisory committees. Muffly: Astellas, Jasper, Adaptive, Baxalta: Research Funding; Adaptive: Honoraria, Other: fees for non-CME/CE services: , Research Funding; Pfizer, Amgen, Jazz, Medexus, Pfizer: Consultancy. OffLabel Disclosure: CD22-directed CAR-T therapy for the treatment of adults with relapsed/refractory large B-cell lymphoma

Abstract: BACKGROUND: CD22 is expressed on the majority of B-cell malignancies. Autologous CAR T-cells targeting CD22 (CAR22) have yielded objective response rates (ORR) of 70-90% in pediatric patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL), including those who had previously failed CD19-directed CAR T-cell (CAR19) therapy. Based on these encouraging results, we evaluated CAR22 in adult patients with R/R ALL and for the first time in patients with R/R large B-cell lymphoma (LBCL), including those who had failed prior autologous CAR19 therapy. METHODS: This single-institution phase I dose escalation clinical trial (NCT04088890) is evaluating a CAR construct incorporating the m971 CD22 single chain variable fragments and 41BB/CD3z endodomains integrated within autologous T-cells via lentiviral transduction. After lymphodepletion with fludarabine and cyclophosphamide, patients were infused with fresh or cryopreserved CAR T-cells after a 7- to 11-day closed manufacturing process utilizing the CliniMACS Prodigy device (Miltenyi). The current cohort includes patients treated at dose level 1 (DL1), which was 1x106 CAR+ cells/kg. Primary objectives assessed the ability to successfully manufacture CAR22 and safety. Overall response rate (ORR) at 28 days post-infusion (D28) was a secondary objective. RESULTS: Three patients with LBCL have been enrolled with a median age of 53 years (range, 51-57) and a median of 6 (range, 5-8) prior lines of therapy. All three patients received prior CAR19 and had refractory disease to second-line or later therapy (n=3); had not undergone autologous hematopoietic stem cell transplantation (HSCT) (n=3); had MYC and BCL2 gene rearrangements (double-hit lymphoma; n=2); had high tumor burden (SPD & gt;50 cm2; n=2); had a history of primary refractory disease (n=1); or had never achieved CR to any therapy (n=1). Six patients with ALL have been enrolled with a median age of 43.5 years (range, 23-62) and a median of 6 (range, 4-8) prior lines of therapy. All six patients received prior allogeneic HSCT and had Ph-positive disease (n=3); had central nervous system (CNS) involvement (n=3); had extramedullary disease (n=2); had high disease burden (BM blasts & gt;5%; n=2); had received prior CD19-directed therapy (n=5); or had received prior CD22-directed therapy (n=3). Successful manufacturing of cells at DL1 was achieved in all patients. All patients (LBCL n=3, ALL n=6) reached day 28 and are included in the safety and response analysis presented here; updated results will be presented at the meeting. Eight patients (88.9%) experienced cytokine release syndrome (CRS); all were Grade 1-2. There were no cases of immune effector cell-associated neurotoxicity syndrome (ICANS). No differences in toxicities were seen across the patient age spectrum and no Grade 5 toxicities occurred following CAR22 infusion. In LBCL, all patients achieved a response at D28 (ORR=100%; CR, n=1, PR, n=2). Both patients with a D28 PR improved to CR by day 90 and 180. All patients remain in CR, with a median follow-up of 8.4 months (range, 6-9.3). In ALL, all patients achieved a CR at D28 (ORR=100%; MRD-, n=5, MRD+, n=1). After a median follow up of 5.1 months (range, 1-8.2), three patients relapsed at 2.5, 4, and 5.5 months after infusion; one patient died while undergoing subsequent therapy 7.3 months post-infusion. CD22 expression by flow cytometry was downregulated or absent in two patients after relapse. Peak CAR expansion as detected by peripheral blood flow cytometry reached a median level of 90.1 (LBCL; range, 85.4-350) and 43.4 (ALL; range, 0.9-399.6) CAR+ cells/µL between D14 and D21. In two LBCL patients with progression following CAR19, CAR22 levels were 11.7 and 55.9 fold higher than prior CAR19 levels at peak expansion. CONCLUSIONS: Infusion of CD22-targeting CAR T-cells in R/R LBCL and ALL is safe and well tolerated. Manufacturing of CAR22 was uniformly successful. To date, 3 of 3 heavily treated adult patients with LBCL whose disease relapsed after prior CAR19 have each achieved CR durable to at least 6 months. All adult ALL patients have achieved CR following CAR22, with some early relapses observed. Accrual is ongoing. Disclosures Negrin: Amgen: Consultancy; Biosource: Current equity holder in private company; UpToDate: Honoraria; KUUR Therapeutics: Consultancy; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company. Rezvani:Pharmacyclics: Research Funding. Shiraz:ORCA BioSystems: Research Funding; Kite, a Gilead Company: Research Funding. Sidana:Janssen: Consultancy. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company. Miklos:Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding. Muffly:Amgen: Consultancy; Adaptive: Research Funding; Servier: Research Funding. OffLabel Disclosure: CD22-directed CAR T-cell Therapy for the treatment of adults with relapsed/refractory LBCL and B-ALL

Abstract: Monitoring of measurable residual disease (MRD) is essential to the management of acute lymphoblastic leukemia (ALL) and is typically performed through repeated bone marrow (BM) assessments. Using a next-generation sequencing (NGS) MRD platform, we performed a prospective observational study evaluating the correlation between peripheral blood (PB) and BM MRD in adults with ALL receiving cellular therapies (hematopoietic cell transplantation [HCT] and chimeric antigen receptor T-cell [CAR-T] therapies). Among the study cohort (N = 69 patients; 126 paired PB/BM samples), we found strong correlation between PB and BM MRD (r = 0.87; P & lt; .001), with a sensitivity and specificity of MRD detection in the PB of 87% and 90%, respectively, relative to MRD in the BM. MRD became detectable in the PB in 100% of patients who subsequently relapsed following HCT, with median time from MRD+ to clinical relapse of 90 days, and in 85% of patients who relapsed following CAR T, with median time from MRD+ to clinical relapse of 60 days. In adult patients with ALL undergoing cellular therapies, we demonstrate strong concordance between NGS-based MRD detected in the PB and BM. Monitoring of ALL MRD in the PB appears to be an adequate alternative to frequent invasive BM evaluations in this clinical setting.

Abstract: Hematopoietic stem cells reside in the bone marrow (BM) niches in the endosteum region, in close proximity to bone-forming osteoblasts and bone-resorbing osteoclasts (Ocl). Tightly regulated SDF-1/CXCR4 interactions retain stem and progenitor cells within their stromal microenvironment. G-CSF induced mobilization impairs this balance by inducing SDF-1 secretion, its proteolytic degradation, and CXCR4 upregulation. Increased Ocl activity in the BM of G-CSF treated mice was documented, however, no active role for Ocl in mobilization was revealed. Ocl secrete the mobilizing chemokines IL-8 and SDF-1, the cytokine HGF, and the metalloprotease MMP-9, which participate in stress-induced mobilization. We hypothesized that in addition to their role in physiological bone remodeling, Ocl are also involved in stem cell mobilization. Ten daily injections of either G-CSF, Pamidronate (Ocl apoptotic drug used to treat osteoporotic patients) or both, led to mobilization of murine progenitor cells, accompanied by Ocl activation in the endosteum region (assayed by TRAP staining for activated Ocl). Interestingly, Pamidronate treatment induced two waves of mobilization on days 5 and 10, correlating with increased levels of TRAP+ multinucleated Ocl in the endosteum and increase in SDF-1 and HGF mRNA in the BM. Stress-inducing conditions, which are also known to mediate bone remodeling, namely LPS stimulation (mimicking bacterial infection) and controlled bleeding (mimicking injury), also triggered progenitor mobilization and CXCR4 upregulation. Of note, LPS-induced mobilization was dependent on functional CXCR4 upregulation and MMP2/9 secretion, was accompanied by Ocl activation and reduction in BM SDF-1 levels. Treatment of primary murine osteoblast/ Ocl precursor cultures with G-CSF, SDF-1 and HGF, increased dose dependently the formation of TRAP+ multinucleated Ocl, suggesting that SDF-1 and HGF are important regulators of the cross talk between Ocl activation and stem cell mobilization. Mobilization was documented in mice receiving five daily injections of either SDF-1 (10ug), or HGF (1.5ug) together with increased levels of Ocl precursors in the blood and BM, as well as number and size of TRAP+ Ocl in the endosteum region. Unexpectedly, Pamidronate also activated SDF-1 expression in cultures of primary mouse osteoblasts, suggesting Ocl activation prior to apoptosis by this drug. Finally, the effect of the major Ocl proteinase, cathepsin K (CTK) as a candidate regulator of stem cell mobilization was investigated. Unexpectedly, recombinant human CTK inactivated human SDF-1 and abolished its chemotactic activity in vitro. This process was completely abrogated by a broad range proteinase inhibitor. We identified N-terminal cleavage and a dose dependent degradation of SDF-1 by CTK. These results suggest that CTK produced and secreted by activated Ocl also participates in the impairment of the steady state homeostatic balance of SDF-1/CXCR4 interactions, inducing local degradation of SDF-1 in the endosteum, thus facilitating stem cell mobilization. Our findings indicate that molecules involved in stem and progenitor cell anchorage, migration, and mobilization are also involved in Ocl activation. In summary, our results add mechanistic insight to the osteoblast/Ocl endosteal interactions and molecular pathways that regulate stem cell mobilization, which is of relevance for pathological stem cell malignancies and clinical mobilization, particularly for patients with poor mobilization.