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Chimeric antigen receptor (CAR)–modified T-cell therapies targeting CD19 represent a new treatment option for patients with relapsed/refractory (R/R) B-cell malignancies. However, CAR T-cell therapy fails to elicit durable responses in a significant fraction of patients. Limited in vivo proliferation and survival of infused CAR T cells are key causes of failure. In a phase 1/2 clinical trial of CD19 CAR T cells for B-cell malignancies (#NCT01865617), low serum interleukin 15 (IL-15) concentration after CAR T-cell infusion was associated with inferior CAR T-cell kinetics. IL-15 supports T-cell proliferation and survival, and therefore, supplementation with IL-15 may enhance CAR T-cell therapy. However, the clinical use of native IL-15 is challenging because of its unfavorable pharmacokinetic (PK) and toxicity. NKTR-255 is a polymer-conjugated IL-15 that engages the entire IL-15 receptor complex (IL-15Rα/IL-2Rβγ) and exhibits reduced clearance, providing sustained pharmacodynamic (PD) responses. We investigated the PK and immune cell PDs in nonhuman primates treated with NKTR-255 and found that NKTR-255 enhanced the in vivo proliferation of T cells and natural killer cells. In vitro, NKTR-255 induced dose-dependent proliferation and accumulation of human CD19 CAR T cells, especially at low target cell abundance. In vivo studies in lymphoma-bearing immunodeficient mice demonstrated enhanced antitumor efficacy of human CD19 CAR T cells. In contrast to mice treated with CAR T cells alone, those that received CAR T cells and NKTR-255 had markedly higher CAR T-cell counts in the blood and marrow that were sustained after tumor clearance, without evidence of persistent proliferation or ongoing activation/exhaustion as assessed by Ki-67 and inhibitory receptor coexpression. These data support an ongoing phase 1 clinical trial of combined therapy with CD19 CAR T cells and NKTR-255 for R/R B-cell malignancies.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T cells are novel therapies showing great promise for patients with relapsed or refractory (R/R) aggressive B-cell non-Hodgkin lymphoma (B-NHL). Single-arm studies showed significant variations in outcomes across distinct CD19 CAR T-cell products. To estimate the independent impact of the CAR T-cell product type on outcomes, we retrospectively analyzed data from 129 patients with R/R aggressive B-NHL treated with cyclophosphamide and fludarabine lymphodepletion followed by either a commercially available CD19 CAR T-cell therapy (axicabtagene ciloleucel [axicel] or tisagenlecleucel [tisacel]), or the investigational product JCAR014 on a phase 1/2 clinical trial (NCT01865617). After adjustment for age, hematopoietic cell transplantation-specific comorbidity index, lactate dehydrogenase (LDH), largest lesion diameter, and absolute lymphocyte count (ALC), CAR T-cell product type remained associated with outcomes in multivariable models. JCAR014 was independently associated with lower cytokine release syndrome (CRS) severity compared with axicel (adjusted odds ratio [aOR], 0.19; 95% confidence interval [CI]; 0.08-0.46), with a trend toward lower CRS severity with tisacel compared with axicel (aOR, 0.47; 95% CI, 0.21-1.06; P = .07). Tisacel (aOR, 0.17; 95% CI, 0.06-0.48) and JCAR014 (aOR, 0.17; 95% CI, 0.06-0.47) were both associated with lower immune effector cell-associated neurotoxicity syndrome severity compared with axicel. Lower odds of complete response (CR) were predicted with tisacel and JCAR014 compared with axicel. Although sensitivity analyses using either positron emission tomography- or computed tomography-based response criteria also suggested higher efficacy of axicel over JCAR014, the impact of tisacel vs axicel became undetermined. Higher preleukapheresis LDH, largest lesion diameter, and lower ALC were independently associated with lower odds of CR. We conclude that CD19 CAR T-cell product type independently impacts toxicity and efficacy in R/R aggressive B-NHL patients.

Post-transplant lymphoproliferative disorder (PTLD) is a leading cause of cancer death in solid organ transplant recipients (SOTRs). Relapsed or refractory (R/R) PTLD portends a high risk of death and effective management is not well established. CD19-targeted CAR-T cell therapy has been utilized, but the risks and benefits are unknown. We report the first case of diffuse large B-cell lymphoma (DLBCL) PTLD treated with lisocabtagene maraleucel and present a systematic literature review of SOTRs with PTLD treated with CD19 CAR-T therapy. Our patient achieved a complete response (CR) with limited toxicity but experienced a CD19

Chimeric antigen receptor T-cells (CAR-T cells) are a new modality of oncological treatment which has demonstrated impressive response in refractory or relapsed diseases, such as acute lymphoblastic leukemia (ALL), lymphomas, and myeloma but is also associated with unique and potentially life-threatening toxicities. The most common adverse events (AEs) include cytokine release syndrome (CRS), neurological toxicities, such as the immune effector cell-associated neurotoxicity syndrome (ICANS), cytopenias, infections, and hypogammaglobulinemia. These may be severe and require admission of the patient to an intensive care unit. However, these AEs are manageable when recognized early and treated by a duly trained team. The objective of this article is to report a consensus compiled by specialists in the fields of oncohematology, bone marrow transplantation, and cellular therapy describing recommendations on the Clinical Centers preparation, training of teams that will use CAR-T cells, and leading clinical questions as to their use and the management of potential complications.

Chimeric antigen receptor T (CAR-T) cell therapy is a novel therapeutic modality for acute lymphoblastic leukemia (ALL) with robust outcomes in patients with refractory or relapsed disease. At the same time, CAR-T cell therapy is associated with unique and potentially fatal toxicities, such as cytokine release syndrome (CRS) and neurological toxicities (ICANS). This manuscript aims to provide a consensus of specialists in the fields of Hematology Oncology and Cellular Therapy to make recommendations on the current scenario of the use of CAR-T cells in patients with ALL.

Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) have produced impressive minimal residual disease–negative (MRD-negative) complete remission (CR) rates in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the factors associated with durable remissions after CAR T-cell therapy have not been fully elucidated. We studied patients with relapsed/refractory B-ALL enrolled in a phase 1/2 clinical trial evaluating lymphodepletion chemotherapy followed by CD19 CAR T-cell therapy at our institution. Forty-five (85%) of 53 patients who received CD19 CAR T-cell therapy and were evaluable for response achieved MRD-negative CR by high-resolution flow cytometry. With a median follow-up of 30.9 months, event-free survival (EFS) and overall survival (OS) were significantly better in the patients who achieved MRD-negative CR compared with those who did not (median EFS, 7.6 vs 0.8 months; P < .0001; median OS, 20.0 vs 5.0 months; P = .014). In patients who achieved MRD-negative CR by flow cytometry, absence of the index malignant clone by IGH deep sequencing was associated with better EFS (P = .034). Stepwise multivariable modeling in patients achieving MRD-negative CR showed that lower prelymphodepletion lactate dehydrogenase concentration (hazard ratio [HR], 1.38 per 100 U/L increment increase), higher prelymphodepletion platelet count (HR, 0.74 per 50 000/μL increment increase), incorporation of fludarabine into the lymphodepletion regimen (HR, 0.25), and allogeneic hematopoietic cell transplantation (HCT) after CAR T-cell therapy (HR, 0.39) were associated with better EFS. These data allow identification of patients at higher risk of relapse after CAR T-cell immunotherapy who might benefit from consolidation strategies such as allogeneic HCT. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

BACKGROUND Little is known about pathogen-specific humoral immunity after chimeric antigen receptor–modified T (CAR-T) cell therapy for B cell malignancies. METHODS We conducted a prospective cross-sectional study of CD19-targeted or B cell maturation antigen–targeted (BCMA-targeted) CAR-T cell therapy recipients at least 6 months posttreatment and in remission. We measured pathogen-specific IgG against 12 vaccine-preventable infections and the number of viral and bacterial epitopes to which IgG was detected (“epitope hits”) using a serological profiling assay. The primary outcome was the proportion of participants with IgG levels above a threshold correlated with seroprotection for vaccine-preventable infections. RESULTS We enrolled 65 children and adults a median of 20 months after CD19- (n = 54) or BCMA- (n = 11) CAR-T cell therapy. Among 30 adults without IgG replacement therapy (IGRT) in the prior 16 weeks, 27 (90%) had hypogammaglobulinemia. These individuals had seroprotection to a median of 67% (IQR, 59%–73%) of tested infections. Proportions of participants with seroprotection per pathogen were comparable to population-based studies, but most individuals lacked seroprotection to specific pathogens. Compared with CD19-CAR-T cell recipients, BCMA-CAR-T cell recipients were half as likely to have seroprotection (prevalence ratio, 0.47; 95% CI, 0.18–1.25) and had fewer pathogen-specific epitope hits (mean difference, –90 epitope hits; 95% CI, –157 to –22). CONCLUSION Seroprotection for vaccine-preventable infections in adult CD19-CAR-T cell recipients was comparable to the general population. BCMA-CAR-T cell recipients had fewer pathogen-specific antibodies. Deficits in both groups support the need for vaccine and immunoglobulin replacement therapy studies. FUNDING Swiss National Science Foundation (Early Postdoc Mobility grant P2BSP3_188162), NIH/National Cancer Institute (NIH/NCI) (U01CA247548 and P01CA018029), NIH/NCI Cancer Center Support Grants (P30CA0087-48 and P30CA015704-44), American Society for Transplantation and Cellular Therapy, and Juno Therapeutics/BMS., In this prospective study, we investigated antibodies against vaccine-preventable infections and other pathogen-specific antibodies in individuals with remission after CAR-T cell therapy for B lineage malignancies.

CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in patients with relapsed/refractory B-cell malignancies; however, it is associated with toxicities including cytokine release syndrome (CRS), neurotoxicity, and impaired hematopoietic recovery. The latter is associated with high-grade cytopenias requiring extended growth factor or transfusional support, potentially leading to additional complications such as infection or hemorrhage. To date, the factors independently associated with hematologic toxicity have not been well characterized. To address this deficit, we retrospectively analyzed 173 patients who received defined-composition CD19 CAR T-cell therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov; NCT01865617), with primary end points of absolute neutrophil count and platelet count at day-28 after CAR T-cell infusion. We observed cumulative incidences of neutrophil and platelet recovery of 81% and 75%, respectively, at 28 days after infusion. Hematologic toxicity was noted in a significant subset of patients, with persistent neutropenia in 9% and thrombocytopenia in 14% at last follow-up. Using debiased least absolute shrinkage selector and operator regression analysis for high-dimensional modeling and considering patient-, disease-, and treatment-related variables, we identified increased CRS severity as an independent predictor for decreased platelet count and lower prelymphodepletion platelet count as an independent predictor of both decreased neutrophil and platelet counts after CD19 CAR T-cell infusion. Furthermore, multivariable models including CRS-related cytokines identified associations between higher peak serum concentrations of interleukin-6 and lower day-28 cell counts; in contrast, higher serum concentrations of transforming growth factor-β1 were associated with higher counts. Our findings suggest that patient selection and improved CRS management may improve hematopoietic recovery after CD19 CAR T-cell therapy.

The description of a so-called cytokine storm in patients with COVID-19 has prompted consideration of anti-cytokine therapies, particularly interleukin-6 antagonists. However, direct systematic comparisons of COVID-19 with other critical illnesses associated with elevated cytokine concentrations have not been reported. In this Rapid Review, we report the results of a systematic review and meta-analysis of COVID-19 studies published or posted as preprints between Nov 1, 2019, and April 14, 2020, in which interleukin-6 concentrations in patients with severe or critical disease were recorded. 25 COVID-19 studies (n=1245 patients) were ultimately included. Comparator groups included four trials each in sepsis (n=5320), cytokine release syndrome (n=72), and acute respiratory distress syndrome unrelated to COVID-19 (n=2767). In patients with severe or critical COVID-19, the pooled mean serum interleukin-6 concentration was 36·7 pg/mL (95% CI 21·6-62·3 pg/mL; I2=57·7%). Mean interleukin-6 concentrations were nearly 100 times higher in patients with cytokine release syndrome (3110·5 pg/mL, 632·3-15 302·9 pg/mL; p

Background Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy which uses genetically modified T cells engineered to express CARs to recognize and kill cells that harbor specific antigens. CAR T-cell products designed to target the tumor specific antigens have been used for the treatment of relapse and/or refractory of acute lymphoblastic leukemia, non-Hodgkin lymphomas, myeloma, and solid tumor in clinical trials at our institution. Several side effects have been reported including increased risk of infection. Methods Retrospective review of morphologic, microbiologic and flow cytometric evaluations done on bronchioalveolar lavages (BAL), pleural effusions, and tissue biopsy specimens from post CAR T-cell adult patients with respiratory complications at our institution from March 2013 to January 2020. Results Thirteen cases with BAL, 8 cases with biopsy (including lymph node and lung tissue) and 5 cases with pleural effusion are reviewed. All infectious diseases were detected on BAL specimens; while primary disease involvements were mainly seen on biopsy and pleural effusion specimens (figure 1). Interestingly, we found a case of a patient with refractory diffuse large B cell lymphoma that had developed mediastinal lymphadenopathy 9 months after CD19 CAR T-cell infusion (defined composition CD4 and CD8 CAR T-cells). Subsequent biopsies showed granulomatous inflammation with minimal evidence of necrosis (figure 2). Special stains with AFB, Gram, Warthin Starry, and Wright Giemsa showed no evidence of infectious organisms. Special stains were negative for acid fast, fungal, bacterial, or spirochetal organisms. Polymerase chain reaction for Mycobacterium tuberculosis complex DNA by hsp65 amplified probe, and nontuberculous mycobacteria (including Mycobacterium avium complex) by 16s rDNA, hsp65, and rpoB pruner sets were both negative. No abnormal B or T cell population was found by concurrent flow cytometry; however, CAR T-cells were detected at low levels. Conclusions Granulomatous inflammation is a chronic, histiocytic response to various chemical mediators of cell injury caused by broad etiologies. δγT cells and T helper cells play roles on recruiting circulating monocytes and maturating of macrophages and ultimately the formation of granulomas. In our case, the patient has no documented autoimmune disease. Extensive infectious disease work-ups failed to identified infectious etiology. The presence of CAR T-cells in mediastinal lymph node 9 months after infusion is not unexpected given CAR T-cells can be detected in the blood for years in some patients. The granulomatous inflammation can be part of exaggerated tissue repair process after lymphoma cells killed by CAR T-cells. However, it may complicate or even mislead assessment of therapeutic effect on post-CAR T-cell cases. The finding of enlarged lymph nodes by imaging study certainly raises concern of disease persistent or relapse. Ethics Approval The study was approved by Fred Hutchinson Cancer Research Center’s Ethics Board, approval numbers 1837, 9364 Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of the this journal.

Background Our institution has treated over 300 patients with chimeric antigen receptor (CAR) T-cell immunotherapy (CAR T-cell therapy) since 2013. Phase I and II trials were primarily based on heavily treated patients with B cell acute lymphoblastic leukemia (B-ALL), aggressive diffuse large B cell lymphoma (DLBCL), and multiple myeloma (MM) who had failed multiple lines of prior chemotherapy and/or hematopoietic stem cell transplantation (HSCT). In these relapsed and/or refractory patients, CAR-T therapy resulted in complete remission in 93% of B-ALL, 60% of DLBCL, and ~80% of MM. Our Pathology Group at Fred Hutch have reviewed and diagnosed various patients with interesting relapse or complications as a result of CAR T-cell therapy. Here we present a retrospective review of autopsies from CAR T-cell therapy. Methods A search for all autopsies conducted on patients from Seattle Cancer Care Alliance/University of Washington Medical Center was performed using the keywords ‘CAR T’ and ‘Chimeric-antigen’. Our inclusion criteria were patients treated with CAR T-cell therapy. Pathology and clinical records were reviewed for cause of death, disease and treatment timelines, microbiology data, cytokine levels, other pathology biopsies, and pertinent laboratory values. Histologic tissues were reviewed. Results Twelve autopsies were performed since 2013. Patient characteristics and causes of death are summarized in table 1. The most common cause of death was due to infectious causes (n=6). Two patients (Patients 10 and 11) had cardiovascular related deaths. Six patients (Patients 1, 2, 6, 7, 10, 12) suffered from CRS in their post-infusion course, four of whom (Patients 1, 2, 7, 10) had CRS directly attributed as the cause of death. CRS was further complicated by immune effector cell-associated neurotoxicity syndrome (ICANS) in 5 patients (Patients 1, 5, 6, 7, and 12). CRS with ICANS was the second most common cause of death in patients treated with CAR T-cells. Three patients (Patients 1, 4, 9) had progression of disease that attributed to cause of death. Conclusions CAR T-cell therapy is a highly effective treatment even for patients who have relapsed and/or refractory disease. Post-therapy complications range in severity and may be fatal in rare instances as in the patients summarized in this study. Infection, CRS with ICANS are the most common causes of death in our single institution study. Ethics Approval The study was approved by Fred Hutchinson Cancer Research Center’s Institutional Review Board, approval number 1837 Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies. Yet, CD19 CAR T cells fail to induce durable responses in most patients. Second infusions of CD19 CAR T cells (CART2) have been considered as a possible approach to improve outcomes. We analyzed data from 44 patients with R/R B-cell malignancies (acute lymphoblastic leukemia [ALL], n = 14; chronic lymphocytic leukemia [CLL], n = 9; non-Hodgkin lymphoma [NHL], n = 21) who received CART2 on a phase 1/2 trial (NCT01865617) at our institution. Despite a CART2 dose increase in 82% of patients, we observed a low incidence of severe toxicity after CART2 (grade ≥3 cytokine release syndrome, 9%; grade ≥3 neurotoxicity, 11%). After CART2, complete response (CR) was achieved in 22% of CLL, 19% of NHL, and 21% of ALL patients. The median durations of response after CART2 in CLL, NHL, and ALL patients were 33, 6, and 4 months, respectively. Addition of fludarabine to cyclophosphamide-based lymphodepletion before the first CAR T-cell infusion (CART1) and an increase in the CART2 dose compared with CART1 were independently associated with higher overall response rates and longer progression-free survival after CART2. We observed durable CAR T-cell persistence after CART2 in patients who received cyclophosphamide and fludarabine (Cy-Flu) lymphodepletion before CART1 and a higher CART2 compared with CART1 cell dose. The identification of 2 modifiable pretreatment factors independently associated with better outcomes after CART2 suggests strategies to improve in vivo CAR T-cell kinetics and responses after repeat CAR T-cell infusions, and has implications for the design of trials of novel CAR T-cell products after failure of prior CAR T-cell immunotherapies.

Background: Autologous T cells engineered to express a CD19 or BCMA-specific chimeric antigen receptor (CAR) have shown high overall response rates in treatment-refractory B-cell non-Hodgkin lymphoma (NHL) and BCMA + multiple myeloma (MM), respectively. However, most patients will eventually relapse, and thus strategies are needed to further improve the efficacy and durability of CAR-T cell products. NKTR-255 is an investigational polyethylene glycol-modified recombinant human IL-15 (rhIL-15) receptor agonist designed to engage the IL-15 pathway to stimulate and expand natural killer (NK) cells and promote the survival and expansion of memory CD8 + T cells. Preclinical data in B-cell lymphoma xenograft models have shown that NKTR-255 enhanced expansion, survival, and anti-tumor activity of human CD19 CAR-T cells. Furthermore, clinical studies have validated that IL-15 augments anti-tumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Here we report pharmacodynamic (PD) analysis of CAR-T cells from relapsed/refractory (R/R) NHL or MM patients who had prior CAR-T therapy and were subsequently enrolled in an ongoing Phase 1 study of NKTR-255. Methods: Patients with R/R NHL or MM who progressed following prior CAR-T therapy were enrolled in a Phase 1 open-label trial evaluating safety, tolerability, and PD after treatment with NKTR-255 (NCT04136756). Patients received single-agent NKTR-255 IV administered every 3 weeks. Peripheral blood (PB) mononuclear cell samples were obtained before and at intervals following NKTR-255 infusion. CAR-T cells were identified by flow cytometry (FCM) using proprietary reagents to detect the CD19-CAR and BCMA-CAR in combination with antibodies to identify CD3 +, CD4 +, and CD8 + T cells. PD data were analyzed for patients with measurable CAR-T cells at baseline; fold change was calculated as treatment with NKTR-255 over baseline (baseline=1). Results: Patient characteristics are shown in Table 1. A total of 6 patients with prior CAR-T/CAR-NK treatment were evaluated for T/ CAR-T cell counts and Ki67 expression to assess proliferation before and after NKTR-255 administration. A total of 3 patients who received NKTR-255, at 1.5 µg/kg (n=1), 4.5 µg/kg (n=1), and 6.0 µg/kg (n=1), had detectable CAR-T cells at baseline. Following NKTR-255 administration, CD3+ CAR-T cell numbers in PB demonstrated a peak average increase of ~1.7-fold (~70% increase) compared to baseline (range 1.4 to 1.8-fold); Table 1. Three patients had low CAR-T/CAR-NK cell counts at baseline and/or post-NKTR-255 treatment. NKTR-255 treatment led to a reversal of the CD4 +:CD8 + CAR-T ratio in 1 patient, with a ~2-fold increase in CD8 + compared with CD4 + CAR-T cells. FCM in PBMC demonstrated an average of ~1.6-fold increase in total CD8 + T-cell with an average 9-fold increase in the percentage of Ki67 +CD8 + T cells in all CAR-T patients following 1 dose of NKTR-255. Among the 6 patients with prior CAR-T/CAR-NK therapy, NKTR-255 was generally well tolerated, with no treatment-related AEs leading to discontinuation, change of dose, or death. Conclusions: Following treatment with NKTR-255, there was an increase in CAR-T-cells as well as a reversal of the CD4 +:CD8 + ratio in 1 patient. Additionally, NKTR-255 induced expansion of total CD8 + cell fraction with an increase of proliferation index of Ki67 in all reported patients, supporting its role in enhancing the expansion and proliferative ability of cytotoxic T cells. Low baseline CAR-T/CAR-NK cell counts observed in patients may be due to longer time intervals between CAR-T infusion and the first dose of NKTR-255. Although preliminary, these data provide promising evidence of CAR-T cell rescue with NKTR-255 administration and represent a potentially novel means of CAR-T augmentation through enhancement of CD8 + T-cells and supports the further evaluation of NKTR-255 and CAR-T therapy as a potential strategy to enhancing the persistence of CAR-T therapy. Additional CAR-T cell patients are being evaluated following NKTR-255 treatment. Ethics approval The study was approved by the institutional review board of each participating site. Trial registration ClinicalTrials.gov NCT04136756 Figure 1 Figure 1. Disclosures Turtle: Century Therapeutics: Consultancy, Other: Scientific Advisory Board; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Amgen: Consultancy; AstraZeneca: Consultancy, Research Funding; TCR2 Therapeutics: Research Funding; Allogene: Consultancy; PACT Pharma: Consultancy; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Asher Bio: Consultancy; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; T-CURX: Other: Scientific Advisory Board; Nektar Therapeutics: Consultancy, Research Funding. Budde: Merck, Inc: Research Funding; Amgen: Research Funding; Astra Zeneca: Research Funding; Mustang Bio: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Roche: Consultancy; Beigene: Consultancy. Patel: Pfizer: Consultancy; Janssen: Consultancy, Research Funding; BMS Celgene: Consultancy, Research Funding; Oncopeptides: Consultancy. Perales: Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Cidara: Honoraria; Equilium: Honoraria; Incyte: Honoraria, Other; Karyopharm: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Merck: Honoraria; Miltenyi Biotec: Honoraria, Other; MorphoSys: Honoraria; Nektar Therapeutics: Honoraria, Other; NexImmune: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Sellas Life Sciences: Honoraria; Servier: Honoraria; Takeda: Honoraria. Cowan: GSK: Consultancy; Bristol Myers Squibb: Research Funding; Nektar: Research Funding; Cellectar: Consultancy; Abbvie: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Harpoon: Research Funding; Sanofi Aventis: Consultancy, Research Funding; Secura Bio: Consultancy. Saeed: Bristol-Myers Squibb Company: Consultancy; sano-aventis U.S.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceutica Products, LP: Consultancy, Other: investigator; Celgene Corporation: Consultancy, Other: investigator; MEI Pharma Inc: Consultancy, Other: investigator; Kite Pharma: Consultancy, Other: investigator; Other-TG therapeutics: Consultancy, Other: investigator; Nektar Therapeutics: Consultancy, Other: research investigator; MorphoSys AG: Consultancy, Membership on an entity's Board of Directors or advisory committees; Other-Epizyme, Inc.: Consultancy; Other-Secura Bio, Inc.: Consultancy; Seattle Genetics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Chavez: Kite/Gilead: Consultancy; Abbvie: Consultancy; Epizyme: Speakers Bureau; Beigene: Speakers Bureau; ADC Therapeutics: Consultancy, Research Funding; Adaptive Biotech: Research Funding; Novartis: Consultancy; Astra Zeneca: Research Funding; Morphosys: Speakers Bureau; karyopharm: Consultancy; Merck: Research Funding. Hirayama: Bristol myers Squibb: Honoraria; Novartis: Honoraria. Janakiram: BMS: Honoraria; Fate: Research Funding; Nektar Therapeutics: Research Funding. Fong: Dendreon: Research Funding; Janssen: Research Funding; Merck: Research Funding; Roche genentech: Research Funding; BMS: Research Funding; Abbvie: Research Funding; Bavarian Nordic: Research Funding. Lee: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Dixit: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wang: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kai: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Marcondes: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Zalevsky: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Shah: Amgen: Consultancy; Janssen: Research Funding; Indapta Therapeutics: Consultancy; Precision Biosciences: Research Funding; Poseida: Research Funding; Nektar: Research Funding; Karyopharm: Consultancy; Sanofi: Consultancy; GSK: Consultancy; CSL Behring: Consultancy; CareDx: Consultancy; BMS/Celgene: Research Funding; Kite: Consultancy; Oncopeptides: Consultancy; Bluebird Bio: Research Funding; Sutro Biopharma: Research Funding; Teneobio: Research Funding.

Introduction Lymphodepletion chemotherapy followed by infusion of autologous T cells engineered to express a CD19-targeted chimeric antigen receptor (CAR) has shown high response rates in relapsed or refractory high-risk chronic lymphocytic leukemia (CLL). Durable remissions were observed, particularly in patients (pts) who achieved minimal residual disease (MRD)-negative (neg) complete response (CR) in the marrow with no detectable malignant clone by IGH sequencing. Here, we present long-term outcomes of CLL pts treated with CD19 CAR-T cell immunotherapy, and single-cell cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analyses of infusion products (IPs) to identify intrinsic T cell characteristics associated with outcomes. Methods Pts were treated on a phase 1/2 clinical trial (NCT01865617) of CD19 CAR-T cell immunotherapy in two cohorts: no ibrutinib (ibr) cohort (No-ibr; ibr discontinued prior to leukapheresis or lymphodepletion) or concurrent ibr cohort (Con-ibr; ibr started at least 2 weeks prior to leukapheresis and continued until at least 3 months after CAR-T cell infusion). Responses were evaluated according to the 2018 International Workshop on CLL (iwCLL) criteria (Hallek et al. Blood 2018). MRD in the marrow was assessed 4 weeks after CAR-T cell infusion using flow cytometry (sensitivity, 10 -4) and IGH sequencing (sensitivity, 10 -6). Multiomics profiling was performed on IPs from 25 pts selected based on distribution of ibr washout time before leukapheresis and clinical response (Table 1). All pts received cyclophosphamide and fludarabine lymphodepletion and 2 x 10 6 CAR-T cells/kg. CD3 + CAR-T cells isolated from IPs underwent CITE-seq (10x Genomics), with generation of gene expression, full-length paired V(D)J, and cell surface protein expression libraries. Differential gene expression (DEG) analyses were performed using Model-based Analysis of Single-cell Transcriptomics (MAST). Blood transcriptome modules were used for Gene Set Enrichment Analysis (GSEA). Results Forty-seven pts (median age, 61 years) with CLL (excluding Richter's transformation without coexisting CLL) received lymphodepletion and CD19 CAR-T cell infusion. Among 29 pts with a pretreatment trackable malignant IGH clone who achieved MRD-neg CR by flow cytometry, at a median follow-up of 23.5 months (interquartile range, 11.0-33.8), the median progression-free survival was 8.5 months (95% confidence interval [CI], 2.9-not reached) for those with a detected post-treatment malignant IGH clone in marrow (n = 11) and could not be estimated (95% CI, 22.2-not reached) in those without a detected post-treatment malignant IGH clone (n = 18; P = .0006; Figure 1A). In the pts without a detected malignant IGH clone 4 weeks after infusion, only 3 (17%) relapsed; 1 died of a myocardial infarction while in ongoing response. We performed multiomics studies on IPs from a selected subset (n = 25) of pts (Table 1) to identify gene expression patterns impacting outcomes. GSEA revealed association with T cell activation, oxidative phosphorylation, transcription initiation and cell cycle modules in IPs of pts who did compared to did not achieve MRD-neg CR (Figure 1B, left), consistent with the hypothesis that CAR-T cell quality is a key determinant of depth and duration of remission in CLL. IPs studied in the multiomics subset were manufactured from pts treated in either the No-ibr or the Con-ibr cohort (Table 1) and our prior data showed a trend towards higher rates of IGH-neg CR in the Con-ibr cohort (85 vs 60%, P = .34). Because ibr administration before leukapheresis might improve CAR-T cell quality and function, we compared IPs of patients in the No-Ibr vs. Con-Ibr cohorts. GSEA revealed that the T cell differentiation via ITK and PKC module was less enriched in IPs of pts in the Con-ibr vs. No-ibr cohort (Figure 1B, right), consistent with ibr-induced inhibition of T cell differentiation as a mechanism for enhanced CAR-T cell quality in the IP. Analyses are ongoing and full results will be presented at the meeting. Conclusion Long-term follow-up confirmed that achieving a malignant IGH-neg response after CAR-T cell therapy is associated with durable remissions in high-risk CLL. Integrated multiomics profiling suggests that CAR-T cell IP quality is a key determinant of MRD-neg CR. Ibr-related inhibition of ITK signaling in T cells can further contribute to the quality of infused CAR-T cells. Figure 1 Figure 1. Disclosures Hirayama: Novartis: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Sheih: Umoja Biopharma: Current Employment. Gauthier: Janssen: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Multerra Bio: Consultancy; Larvol: Consultancy; JMP: Consultancy; Eusapharma: Consultancy. Maloney: A2 Biotherapeutics: Divested equity in a private or publicly-traded company in the past 24 months; BioLineRx, Juno, Celgene, Kite, a Gilead Company, Gilead, Novartis, and Pharmacyclics: Honoraria; A2 Biotherapeutics: Consultancy; Kite, a Gilead Company, Juno, and Celgene: Research Funding; Juno: Patents & Royalties. Gottardo: Illumina: Consultancy; 10x Genomics: Current holder of stock options in a privately-held company; Ozette Technologies: Current holder of individual stocks in a privately-held company; Modulus Therapeutics: Current holder of individual stocks in a privately-held company. Turtle: PACT Pharma: Consultancy; TCR2 Therapeutics: Research Funding; Nektar Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; AstraZeneca: Consultancy, Research Funding; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Asher Bio: Consultancy; Century Therapeutics: Consultancy, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Allogene: Consultancy; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; T-CURX: Other: Scientific Advisory Board.

Background CD19-directed chimeric antigen receptor (CAR)-modified T cell therapy expansion and persistence in peripheral blood has been shown to correlate with responses against B cell malignances (Hay, Blood, 2018; Ayuk, Blood Adv, 2021). CAR-T cell enumeration is typically achieved through quantitative PCR (qPCR) or flow cytometry (FC). qPCR, however, requires techniques that may not be locally validated and, unlike FC, cannot simultaneously address T cell immunophenotype. Existing FC reagents to detect CD19 CAR-T cells are mostly specific for a given CAR sequence or bind non-specifically to the introduced protein (e.g. Protein A). Utilizing glycosylation data from the structure of CD19(Teplykov, Proteins, 2018), we created a stable, biotinylated CD19 ectodomain glycomutant reagent (gmCD19) that binds to the CD19-directed scFv in CAR constructs and can be detected by FC. Methods The gmCD19 ectodomain (amino acids 21-227, N138Q) was expressed in 293 Freestyle™ cells with a C-terminal 6x Histidine-AviTag™ to facilitate expression, purification, FC detection (with fluorophore-bound streptavidin) and enable tetramer formation. We used FC to determine limit of detection (LoD), defined as the percent of detectable CAR-T cells when spiked into peripheral blood mononuclear cells (PBMC) at known concentrations. We used a CD19-directed CAR-T cell that co-expresses truncated EGFR (EGFRt) to benchmark gmCD19 and compare our reagent with commercially available detection reagents. This was quantitated by Pearson correlation coefficient and Bland-Altman measure of bias - defined as the mean of differences between tests on the same sample. We also tested reagent stability by assessing for decrease in mean fluorescence intensity (MFI) and the percent of CAR-T cells detected after multiple freeze-thaw cycles of the gmCD19 reagent. We compared CAR-T cell detection by gmCD19 with qPCR from actual patient samples treated with axicabtagene ciloleucel (axi-cel) and collected into three different anticoagulants. Results gmCD19 detected as few as 0.25% CAR-T cells by FC and was highly correlated with EGFRt expression (r=0.9993, p Conclusion The availability of multiple CD19-directed CAR-T cell products and the importance of monitoring CAR-T cell expansion and persistence in patients undergoing CAR-T cell therapy creates the necessity for an easily applied, stable, and reliable quantitation FC method. We show that our gmCD19 accurately measures CD19-directed CAR-T cells across a variety of CAR-T cell constructs, including commercially available products. Disclosures Sheih: Umoja Biopharma: Current Employment. Fiorenza: Bristol Myers Squibb: Research Funding; Link Immunotherapeutics: Consultancy. Hirayama: Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Honoraria. Chou: Genentech: Current Employment. Gauthier: Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Multerra Bio: Consultancy; Larvol: Consultancy; JMP: Consultancy; Eusapharma: Consultancy. Correnti: Link Immunotherapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Turtle: PACT Pharma: Consultancy; Amgen: Consultancy; Asher Bio: Consultancy; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; T-CURX: Other: Scientific Advisory Board; Century Therapeutics: Consultancy, Other: Scientific Advisory Board; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Nektar Therapeutics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; TCR2 Therapeutics: Research Funding; Allogene: Consultancy.

Background: Chimeric antigen receptor-engineered (CAR) T-cell therapy remains associated with significant toxicities including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Recently, the recombinant IL-1 receptor antagonist anakinra has emerged as a promising approach after failure of tocilizumab and corticosteroids to treat CRS/ICANS (Norelli, Nat Med 2018; Giavridis, Nat Med 2018). Here, we describe the safety and efficacy of two anakinra dose regimens to treat refractory CRS and/or ICANS after CAR T-cell therapy. Methods: We retrospectively analyzed data from 26 patients with B-cell or plasma cell malignancies treated at 9 institutions with anakinra for CRS and/or ICANS after CAR T-cell therapy. Details regarding CAR T-cell product and disease type are shown in the Table. CRS/ICANS grade was determined by applying the ASTCT criteria at the time of peak symptom severity. We defined response to anakinra as an improvement in CRS and/or ICANS symptoms per the attending physician's evaluation. Results: Patients, disease, and CAR T-cell product are shown in the Table. Anakinra was administered at 100-200mg/day subcutaneously (SC) in 13 patients (pts) (50%; low-dose), or at 8mg/kg/day SC or intravenously (IV) in 13 pts (50%; high-dose). Most pts were treated with anakinra for steroid-refractory ICANS (n=23); two pts were treated for tocilizumab-refractory CRS (n=2) and one for both (n=1). All but one patient received anakinra concurrently with corticosteroids. Median peak CRS and ICANS grade by ASTCT criteria was 2 (range, 1-4), and 4 (range, 0-5), respectively. Median CRS and ICANS duration was 5 days (range, 1-10) and 15.5 days (range, 1-38), respectively. Median time from CAR T-cell infusion to anakinra initiation was 9 days (range, 5-31). The median duration of anakinra treatment was 8.5 days (range, 1-47). The median time to anakinra initiation from CRS or ICANS onset was comparable in pts receiving high-dose compared to low-dose anakinra (4 versus 4 days, respectively; p=0.8). Comparable peak CRS (median grade, 2 versus 2, p=0.9) and ICANS (median grade, 4 versus 4, p=0.2) were measured in both groups. Other toxicity-directed therapies were administered in 8 pts receiving low-dose anakinra (siltuximab, n=8; intrathecal chemotherapy, n=2, etoposide n=1). The only infectious event reported after anakinra initiation was HHV6 encephalitis (n=1). Two pts with infections confirmed prior to anakinra initiation died after anakinra treatment: CMV pneumonia (n=1), Escherichia coli bacteremia (n=1). In one patient the anakinra administration route was changed from SC to IV due to a subcutaneous hematoma; in one patient anakinra was discontinued due to elevated liver enzymes. We observed anti-tumor responses (partial or complete) to CAR T-cell therapy in 15 pts (58%; B-ALL, n=1/1; DLBCL, n=9/15; MCL, n=3/4; MM; n=1/1; PMBCL, n=1/3), including complete responses in 11 pts (42%). In high-dose anakinra pts, the ORR was 77% (complete response, 53%). CRS/ICANS improvement was observed after anakinra initiation in 73% of pts with a median duration of treatment of 3 days (range 1-7). Higher response rates were seen in pts who received high-dose compared to low-dose anakinra (100% versus 46%, respectively; p=0.005) and the non-relapse mortality rate at day 30 was significantly lower in pts treated with high-dose anakinra compared to low-dose anakinra (0% versus 69%; p=0.001%). In addition, a shorter time to anakinra initiation from CRS or ICANS onset was associated with CRS/ICANS improvement (median, 2 versus 5 days in responders versus non-responders, respectively; p=0.04). Conclusion After failure of tocilizumab and/or corticosteroids, early administration of high-dose anakinra (8mg/kg/day IV or SC) was associated with rapid resolution of CRS/ICANS symptoms after use of tocilizumab and/or corticosteroids, with a manageable toxicity profile, and with a non-relapse mortality rate at day 30 of 0%. In contrast, 38% of patients treated with low-dose anakinra died from infections. We observed complete responses to CAR T-cell therapy in pts treated with high-dose anakinra, suggesting limited impact on in vivo CAR-T cell function. In summary, high-dose anakinra is a feasible and promising approach after failure of conventional CRS and ICANS-directed therapies. Prospective trials of anakinra to prevent or treat CRS and ICANS are ongoing. Figure 1 Figure 1. Disclosures Barba: Amgen, Celgene, Gilead, Incyte, Jazz Pharmaceuticals, MSD, Novartis, Pfizer and Roche, Jazz Phar,aceuticals: Honoraria; Cqrlos III heqlth Institute, aSOCIACION espanola contra el cancer, PERIS: Research Funding. Iacoboni: BMS/Celgene, Gilead, Novartis, Janssen, Roche: Honoraria. Kwon: Novartis, Celgene, Gilead, Pfizer: Consultancy, Honoraria. Bailen: Gilead, Pfizer: Speakers Bureau. Reguera: Janssen, Kite/Gilead, Novartis: Speakers Bureau; BMS-Celgene, Novartis: Membership on an entity's Board of Directors or advisory committees. Corral: Gilead: Consultancy; Novartis: Consultancy; Gileqd: Honoraria. Ortiz-Maldonado: Kite, Novartis, BMS, Janssen: Honoraria. Maziarz: Allovir: Consultancy, Research Funding; Novartis: Consultancy, Other: Data and Safety Monitoring board, Research Funding; Vor Pharma: Other: Data and Safety Monitoring Board; Incyte Corporation: Consultancy, Honoraria; Bristol-Myers, Squibb/Celgene,, Intellia, Kite: Honoraria; Artiva Therapeutics: Consultancy; CRISPR Therapeutics: Consultancy; Omeros: Research Funding; Intellia: Honoraria; Athersys: Other: Data and Safety Monitoring Board, Patents & Royalties. Shadman: Mustang Bio, Celgene, Bristol Myers Squibb, Pharmacyclics, Gilead, Genentech, Abbvie, TG Therapeutics, Beigene, AstraZeneca, Sunesis, Atara Biotherapeutics, GenMab: Research Funding; Abbvie, Genentech, AstraZeneca, Sound Biologics, Pharmacyclics, Beigene, Bristol Myers Squibb, Morphosys, TG Therapeutics, Innate Pharma, Kite Pharma, Adaptive Biotechnologies, Epizyme, Eli Lilly, Adaptimmune , Mustang Bio and Atara Biotherapeutics: Consultancy. Green: Seagen Inc.: Research Funding; bristol myers squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; Janssen Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Chow: ADC Therapeutics: Current holder of individual stocks in a privately-held company, Research Funding; AstraZeneca: Research Funding. Hirayama: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Maloney: Kite, a Gilead Company, Juno, and Celgene: Research Funding; A2 Biotherapeutics: Consultancy; BioLineRx, Juno, Celgene, Kite, a Gilead Company, Gilead, Novartis, and Pharmacyclics: Honoraria; A2 Biotherapeutics: Divested equity in a private or publicly-traded company in the past 24 months; Juno: Patents & Royalties. Turtle: AstraZeneca: Consultancy, Research Funding; Nektar Therapeutics: Consultancy, Research Funding; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Century Therapeutics: Consultancy, Other: Scientific Advisory Board; T-CURX: Other: Scientific Advisory Board; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Asher Bio: Consultancy; Amgen: Consultancy; PACT Pharma: Consultancy; TCR2 Therapeutics: Research Funding; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; Allogene: Consultancy. Gauthier: Janssen: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Multerra Bio: Consultancy; Larvol: Consultancy; JMP: Consultancy; Eusapharma: Consultancy.

The description of a so-called cytokine storm in patients with COVID-19 has prompted consideration of anti-cytokine therapies, particularly interleukin-6 antagonists. However, direct systematic comparisons of COVID-19 with other critical illnesses associated with elevated cytokine concentrations have not been reported. In this Rapid Review, we report the results of a systematic review and meta-analysis of COVID-19 studies published or posted as preprints between Nov 1, 2019, and April 14, 2020, in which interleukin-6 concentrations in patients with severe or critical disease were recorded. 25 COVID-19 studies (n=1245 patients) were ultimately included. Comparator groups included four trials each in sepsis (n=5320), cytokine release syndrome (n=72), and acute respiratory distress syndrome unrelated to COVID-19 (n=2767). In patients with severe or critical COVID-19, the pooled mean serum interleukin-6 concentration was 36·7 pg/mL (95% CI 21·6-62·3 pg/mL; I

Background Chimeric antigen receptor-modified T (CAR-T) cell immunotherapy for B cell hematologic malignancies results in prolonged B cell depletion. Little is known about the effects of CAR-T cell therapy on pre-existing pathogen-specific humoral immunity. Methods We conducted a prospective, cross-sectional study of children and adults treated with CD19- or BCMA-CAR-T cell therapy. Eligible patients were ≥ 6 months post-CAR-T cell infusion and in remission without subsequent chemoimmunotherapy. We measured total immunoglobulin G (IgG), pathogen-specific IgG levels for 12 vaccine-preventable infections, and B cell subsets from blood. Seroprotective antibody titers were based on standard thresholds. We described the proportion of patients with seroprotective titers and tested for associations between clinical factors and seroprotection using generalized estimating equations. Results We enrolled 65 patients who received CD19- (n=54) or BCMA- (n=11) CAR-T cell therapy. Seven patients were < 18 years old. Samples were collected a median of 20 months (range, 7–68) after CAR T cell infusion. Seroprotection to vaccine-preventable pathogens was generally comparable to the U.S. population (Fig 1) even though blood CD19+ B cell counts were low (< 20 cells/mm3) in 60% of patients. Among 30 patients without IgG replacement in the prior 16 weeks (4 half-lives of IgG), 27 (90%) had hypogammaglobulinemia. Despite this, these individuals had seroprotection to a median of 67% (IQR, 59%-73%) of tested pathogens (Fig 2A). The proportion of patients with seroprotection was lowest for mumps, hepatitis A and B, H. influenzae type B (Hib), S. pneumoniae, and B. pertussis. Patients receiving BCMA-CAR-T cells had seroprotection to fewer pathogens than those receiving CD19-CAR-T cells (Fig 2B), but the difference did not reach statistical significance (Fig 3). There were no significant differences by other variables. Figure 1. Proportion of CAR-T cell recipients with seroprotection to vaccine-preventable infections compared to the U.S. population, stratified by receipt of IgG replacement in the previous 16 weeks. Figure 2 A-B. Percentage of pathogens with seroprotective antibody titers among patients without IgG replacement in the previous 16 weeks. Figure 3. Association of clinical factors with seroprotection to vaccine-preventable infections among patients without IgG replacement in the previous 16 weeks (n=30) Conclusion Seroprotection for vaccine-preventable infections after CD19-CAR-T cell therapy was comparable to the general population. BCMA-CAR-T cell recipients may benefit most from replacement IgG. Vaccinations after CAR-T cell therapy should be considered and prioritized for S. pneumoniae, Hib, hepatitis viruses, and B. pertussis. Disclosures Justin J. Taylor, PhD, Vir Biotechnology (Grant/Research Support) Damian J. Green, MD, Cellectar Biosciences (Grant/Research Support)GSK (Advisor or Review Panel member)Juno Therapeutics (Grant/Research Support, Advisor or Review Panel member, Other Financial or Material Support, Royalities)Seattle Genetics (Grant/Research Support, Advisor or Review Panel member) Michael Boeckh, MD PhD, AlloVir (Consultant)EvrysBio (Advisor or Review Panel member, Other Financial or Material Support, share options)Gilead (Consultant, Grant/Research Support)GSK (Consultant)Helocyte (Advisor or Review Panel member, Shareholder)Lophius (Grant/Research Support)Merck (Consultant, Grant/Research Support)SymBio (Consultant)VirBio (Consultant, Grant/Research Support) David G. Maloney, MD, PhD, A2 Biotherapeutics (Consultant, Other Financial or Material Support, Stock Options)Bioline Rx (Consultant)Celgene (Consultant, Grant/Research Support)Gilead (Consultant)Juno Therapeutics (Consultant, Research Grant or Support, Other Financial or Material Support, four pending patents, not issued, licensed, no royalities, no licensees)Kite Pharma (Consultant, Grant/Research Support)Novartis (Consultant)Pharmacyclics (Consultant) Cameron J. Turtle, MBBS, PhD, Allogene (Other Financial or Material Support, Ad hoc advisory board (last 12 months))ArsenalBio (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)AstraZeneca (Grant/Research Support, Other Financial or Material Support, Ad hoc advisory board (last 12 months))Caribou Biosciences (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)Century Therapeutics (Advisor or Review Panel member)Eureka Therapeutics (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)Juno Therapeutics (Grant/Research Support, Other Financial or Material Support, Patent: Licensed to Juno Therapeutics)Myeloid Therapeutics (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)Nektar Therapeutics (Grant/Research Support, Other Financial or Material Support, Ad hoc advisory board (last 12 months))PACT Pharma (Other Financial or Material Support, Ad hoc advisory board (last 12 months))Precision Biosciences (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)TCR2 Therapeutics (Grant/Research Support)T-CURX (Advisor or Review Panel member) Joshua A. Hill, MD, Allogene (Consultant)Allovir (Consultant)Gilead (Consultant)Karius (Grant/Research Support, Scientific Research Study Investigator)Takeda (Grant/Research Support, Scientific Research Study Investigator)

Background: CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies, but lack of durable complete response (CR) is still seen in most patients. Enhancing in vivo T cell function or "fitness" is currently an area of intense focus to improve the anti-tumor efficacy of CAR T cell therapy. Supporting this notion, in vivo expansion and persistence of CAR T cells have been associated with better outcomes after CD19 CAR T cell therapy. Homeostatic cytokines, such as IL-15, play a key role in maintaining T cell fitness and formation of long-term immunologic memory. Since the impact of IL-15 on CAR T-cell function and anti-tumor efficacy in humans is not well characterized, we applied multivariable regression modeling to study the relationships between IL-15 serum concentrations, anti-tumor efficacy, and in vivo CAR T cell kinetics using data from 192 patients undergoing CD19 CAR T-cell therapy. Methods: We included patients enrolled on a phase I/II clinical trial (NCT01865617) at our institution who received defined-composition CD19 CAR T cell therapy. Responses were defined according to the 2019 NCCN guidelines for ALL (multiparameter flow-negative marrow CR or CR/PR by PET or CT in the absence of marrow disease), 2018 iwCLL for CLL (CR or PR), and the 2014 Lugano criteria for NHL (CR or PR). Logistic regression and proportional odds logistic regression were used for multivariable modeling of response and CAR T cell kinetics, respectively. In addition to the IL-15 serum concentration, all models included the following predictors: disease type, pre-lymphodepletion (LD) LDH serum concentration, and LD type (cyclophosphamide and fludarabine [CyFlu] versus other). Results: Sixty-four ALL (33%), 49 CLL (25%), and 79 NHL (42%) patients were included. Cyclophophamide and fludarabine-based LD was used in 163 patients (85%). We observed responses to CD19 CAR T cell therapy in 41 of 52 ALL (82%), 32 of 47 CLL (71%), and 38 of 75 NHL patients (54%). Models including pre-LD IL-15 identified interaction effects with disease type (p=0.008), and with pre-LD LDH (p=0.09). The impact of pre-LD IL-15 on response was differentially affected by the pre-LD LDH concentrations across disease types in a multivariable analysis including interaction terms (Fig. A). Adjusting for a pre-LDH value of 333U/L (third quartile), an increase in the pre-LD IL-15 concentration from 2.6 to 16.2pg/µL (1st to 3rd quartile) was associated with a 13.54, 5.61, and 1.33 increase in the odds of response in ALL, CLL, and NHL, respectively. We did not identify interaction effects when considering the day 0, and peak IL-15, or the IL-15 AUCpre-LD-30. Peak IL-15 was strongly associated with response (OR=4.46 per log10 pg/µL increment, p=0.02), with a larger effect size compared to day 0 IL-15 (OR=2.46, p=0.15), and the IL-15 AUCpre-LD-30 (OR=2.87, p=0.05). Higher peak IL-15 was associated with higher response probabilities across all diseases adjusting for the pre-LD LDH concentration (Fig. B). Next, we applied multivariable modeling to predict the CAR transgene AUC between day 0 and day 90 (AUC0-90). We confirmed that higher IL-15 serum concentrations were independently associated with higher CAR transgene AUC0-90; we found a stronger association between CAR transgene AUC0-90 and the peak IL-15 serum concentration (OR=4.71, p Conclusion: Our multivariable analyses show that higher IL-15 serum concentrations are associated with higher probabilities of response and higher CAR transgene expansion and persistence. We estimated distinct relationships between the pre-LD IL-15 serum concentrations and response across disease types. More so in ALL and CLL patients than in NHL patients, high pre-LD IL-15 was associated with efficacy, even in those with high pre-LD LDH concentrations. This suggests that additional factors, such as the tumor microenvironment, might contribute to CAR T cell failure in some NHL patients. Peak IL-15 was independently associated with higher response probabilities across all diseases. Along with the known biological effects of IL-15 on T cell fitness, our findings suggest high levels of IL-15 could improve CD19 CAR T cell efficacy and in vivo persistence. Figure 1 Disclosures Gauthier: JMP, Eusapharma, Multerra Bio: Honoraria. Chou:Nektar Therapeutics: Patents & Royalties. Riddell:Lyell Immunopharma: Other: Founder; Adaptive Biotechnologies, Nohla: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics, A Celgene/BMS Company: Consultancy, Current equity holder in publicly-traded company. Maloney:Bioline Rx: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties: Patents are pending, but not issued, licensed, no royalties, no licensees., Research Funding; MorphoSys: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria. Turtle:Novartis: Consultancy; Humanigen: Consultancy; Kite/Gilead: Consultancy; Allogene: Consultancy; PACT Pharma: Consultancy; Physician Education Resource: Consultancy; AstraZeneca: Consultancy, Research Funding; Century Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Arsenal Bio: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno/BMS: Patents & Royalties, Research Funding; Nektar Therapeutics: Consultancy, Research Funding; Precision Biosciences: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Background Chimeric antigen receptor T-cell (CAR-T) therapy has shown promising efficacy in high-risk CLL but progressive disease after CAR-T is not uncommon. We studied outcomes of pts with relapsed or refractory CLL after CAR-T to establish a benchmark for trials in this setting. Methods CLL pts treated with CD19-specific CAR-T on a clinical trial (NCT01865617) were reviewed. We identified pts with stable or progressive disease (SD/PD) on initial assessment or with relapse after a complete or partial response (CR/PR). Multivariable models included variables related to disease, treatment and CAR-T procedure. Results 28 pts had SD/PD (n=16; 57%) or relapsed (n=12; 43%) disease after CAR-T. Five pts (18%) had prior allo-SCT. Five pts (18%) received bridging therapy after leukapheresis. CAR-T adverse events included CRS in 22 pts (grade 3 in 2) and NT in 7 (grade 3 in 5). 5 of 11 pts who received Venetoclax (Ven) after CAR-T had progressed on it before but had a median duration of response (DOR) of 5 months (2-8) which was not different from pts who were Ven responsive before CAR-T [5 months (1-10); p = 0.95). This was not true for Ibrutinib (IB) with DOR of 2 months (0.5-7) in pre-CAR-T IB failed vs. 12.25 months (6-18.5) in pre-CAR-T IB responsive pts (p = 0.001). Fourteen pts (51%) had repeat CAR-T therapy (CR=3, PR=3) with DOR after second CAR-T of 5.5 months (1-33). Six pts (23%) received an allo-SCT median 6.5 months after CAR-T progression (CR=2, PR=1). History of progression on both IB and Ven before CAR-T was strongly associated with poor OS [hazard ratio (HR) 11.2 (95% CI: 2.5-50.5); p=0.002]. Receiving an allo-SCT after CAR-T progression was associated with an improved OS [HR 0.14 (95% CI: 0.02-0.97); p=0.04]. [Fig-1] Conclusion This data sets a benchmark for clinical trials that intend to improve outcome of CLL pts with progression after CAR-T. OS after CAR-T progression was significantly shorter in pts who received CAR-T after failing both IB and Ven compared to others. This finding supports referring high-risk CLL pts for CAR-T treatment after progression on IB and while still responsive to Ven. For eligible pts, allo-SCT seems to provide a higher chance of survival in pts who progress after CAR-T.

Background CD19-targeted chimeric antigen receptor-engineered (CD19 CAR)-T cell immunotherapy has shown efficacy for relapsed or refractory (R/R) B-cell malignancies, but absence of complete response or relapses are still observed in a significant portion of patients (pts). Objectives To evaluate outcomes after repeat infusion of CD19 CAR-T cells, and identify the factors associated with response, CAR-T cell in vivo expansion, and progression-free survival (PFS). Methods We analyzed data from pts with R/R B-cell malignancies who received a second infusion of CD19 CAR-T cells (CART2) on a phase 1/2 trial (NCT01865617) at our institution. Responses were evaluated 4 weeks after CAR-T cell infusion and defined according to the 2018 NCCN guidelines for acute lymphoblastic leukemia (ALL), 2018 iwCLL for chronic lymphocytic leukemia (CLL), and the Lugano criteria for non-Hodgkin lymphoma (NHL). Results Forty-four pts evaluable for response were included in this study (see Table). Pts were heavily pre-treated (median prior therapies, 6), and 16 pts (36%) had bulky (e" 5cm) disease. Prior to CART2, 15 pts (32%) had not responded to CART1, 22 (50%) relapsed or progressed after initial response (complete response [CR], n=15; partial response [PR], n=7) to CART1, and 7 (16%) were in ongoing PR. We observed responses to CART2 in 3 of 14 ALL pts (21%; all CR/CRi), 4 of 11 CLL pts (36%; CR/CRi, n=3; partial response [PR], n=1), and 9 of 19 NHL pts (47%; CR, n=2; PR, n=7). After a 43-month median follow-up, the estimated 4-year PFS and OS probabilities in responders were 23% (95% CI, 9-59%) and 36% (95% CI 19-71%), respectively. Multivariable logistic regression identified lymphodepletion intensity before CART1 (high-intensity cyclophosphamide and fludarabine [CyFlu] vs Other, OR=12.19, p=0.04; high intensity versus low intensity CyFlu, OR=3.83, p=0.08) and the peak blood CAR-T cell count after CART2 (OR=2.31 per log10 CD8+ CAR-T cell/µL increase, p=0.01) as predictors of response to CART2. In a multivariable Cox model, higher peak CD8+ CAR-T cell count after CART2 (HR=0.47 per log10 CD8+ CAR-T cell/µL increase, p Conclusion Outcomes after second infusions of CD19 CAR-T cells might be improved with high-intensity CyFlu lymphodepletion prior to CART1 and by increasing the CAR-T cell dose at the time of CART2.

We previously reported durable responses in relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) patients treated with CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell immunotherapy after ibrutinib failure. Because preclinical studies showed that ibrutinib could improve CAR T cell-antitumor efficacy and reduce cytokine release syndrome (CRS), we conducted a pilot study to evaluate the safety and feasibility of administering ibrutinib concurrently with CD19 CAR T-cell immunotherapy. Nineteen CLL patients were included. The median number of prior therapies was 5, and 17 patients (89%) had high-risk cytogenetics (17p deletion and/or complex karyotype). Ibrutinib was scheduled to begin ≥2 weeks before leukapheresis and continue for ≥3 months after CAR T-cell infusion. CD19 CAR T-cell therapy with concurrent ibrutinib was well tolerated; 13 patients (68%) received ibrutinib as planned without dose reduction. The 4-week overall response rate using 2018 International Workshop on CLL (iwCLL) criteria was 83%, and 61% achieved a minimal residual disease (MRD)-negative marrow response by IGH sequencing. In this subset, the 1-year overall survival and progression-free survival (PFS) probabilities were 86% and 59%, respectively. Compared with CLL patients treated with CAR T cells without ibrutinib, CAR T cells with concurrent ibrutinib were associated with lower CRS severity and lower serum concentrations of CRS-associated cytokines, despite equivalent in vivo CAR T-cell expansion. The 1-year PFS probabilities in all evaluable patients were 38% and 50% after CD19 CAR T-cell therapy, with and without concurrent ibrutinib, respectively (P = .91). CD19 CAR T cells with concurrent ibrutinib for R/R CLL were well tolerated, with low CRS severity, and led to high rates of MRD-negative response by IGH sequencing.

Patients with follicular lymphoma (FL) with early relapse after initial chemoimmunotherapy, refractory disease, or histologic transformation (tFL) have limited progression-free and overall survival. We report efficacy and long-term follow-up of 21 patients with relapsed/refractory (R/R) FL (n = 8) and tFL (n = 13) treated on a phase 1/2 clinical trial with cyclophosphamide and fludarabine lymphodepletion followed by infusion of 2 × 106 CD19-directed chimeric antigen receptor–modified T (CAR-T) cells per kilogram. The complete remission (CR) rates by the Lugano criteria were 88% and 46% for patients with FL and tFL, respectively. All patients with FL who achieved CR remained in remission at a median follow-up of 24 months. The median duration of response for patients with tFL was 10.2 months at a median follow-up of 38 months. Cytokine release syndrome occurred in 50% and 39%, and neurotoxicity in 50% and 23% of patients with FL and tFL, respectively, with no severe adverse events (grade ≥3). No significant differences in CAR-T cell in vivo expansion/persistence were observed between FL and tFL patients. CD19 CAR-T cell immunotherapy is highly effective in adults with clinically aggressive R/R FL with or without transformation, with durable remission in a high proportion of FL patients. This trial was registered at clinicaltrials.gov as #NCT01865617.

CD19-targeted chimeric antigen receptor-modified T cell (CAR-T cell) therapy has shown excellent antitumor activity in patients with relapsed/refractory B cell malignancies, with very encouraging response rates and outcomes. However, the late effects following this therapy remain unknown. Here we report late adverse events-defined as starting or persisting beyond 90 days after CAR-T cell infusion-in patients who survived at least 1 year after therapy. The median duration of follow-up was 28.1 months (range, 12.5 to 62.6 months). At last follow-up, 73% of patients were still alive and 24% were in ongoing complete remission (CR). The most common late adverse event was hypogammaglobulinemia (IgG400 mg/dL or i.v immunoglobulinm (IVIG) replacement, observed in 67% of the patients with available data. Infection density was .55 infection/100 days at risk (2.08 per patient-year). The majority (80%) of the infections were treated in the outpatient setting, and 5% necessitated admission to the intensive care unit (ICU). Subsequent malignancies occurred in 15% of patients, including 5% with myelodysplastic syndrome (MDS). Among patients with ongoing CR and with no MDS, 16% experienced prolonged cytopenia requiring transfusions or growth factor support. Graft-versus-host disease occurred in 3 of 15 patients (20%) who had undergone previous allogeneic hematopoietic cell transplantation. Most of the late events observed in this cohort were not severe, and many could be related to previous or subsequent therapies, suggesting a safe long-term profile of CD19-targeted CAR-T cell immunotherapy.

Allogeneic hematopoietic cell transplantation (allo-HCT) is offered to selected patients after chimeric antigen receptor-modified T-cell (CAR-T) therapy. Lymphodepleting chemotherapy and CAR-T therapy have immunosuppressive and immunomodulatory effects that could alter the safety profile of subsequent allo-HCT. We reviewed our experience with 32 adults (acute lymphoblastic leukemia [ALL], n = 19; B-cell non-Hodgkin lymphoma [NHL]/chronic lymphocytic leukemia [CLL], n = 13) who received an allo-HCT after CAR-T therapy, with a focus on posttransplant toxicities. Myeloablative conditioning (MAC) was used in 74% of ALL patients and 39% of NHL/CLL patients. The median time from CAR-T therapy to allo-HCT was 72 days in ALL patients and 122 days in NHL/CLL patients. Cumulative incidences of grade 3-4 acute graft-versus-host disease (GVHD) and chronic GVHD were 25% and 10%, respectively. All patients had neutrophil recovery (median, 18.5 days) and all but 3 had platelet recovery (median, 12 days). Twenty-two percent had viral or systemic fungal infection within 100 days after allo-HCT. The 100-day and 1-year cumulative incidences of NRM were 16% and 21%, respectively, for ALL patients and 15% and 33%, respectively, for NHL/CLL patients. In ALL patients, later utilization of allo-HCT after CAR-T therapy was associated with higher mortality. In NHL/CLL patients, MAC was associated with higher mortality. Toxicities did not exceed the expected incidences in this high-risk population.

Introduction Despite promising results in patients with relapsed/refractory B cell malignancies, CD19 CAR-T cell therapy can be associated with significant toxicities. Specifically, impaired hematopoietic recovery can be observed in a subset of patients. Objectives We aimed to describe hematopoietic recovery following CD19 CAR-T cell therapy, and to identify factors independently associated with this complication using multivariable modeling. Methods We retrospectively analyzed 125 adult patients with R/R acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia (CLL), treated with CD19 CAR-T cells on a phase 1/2 clinical trial (characteristics summarized in Table 1). Criteria for neutropenia, thrombocytopenia, and recovery were defined as per the CIBMTR reporting guidelines. For competing risk analysis, an event was defined as ANC or platelet (Plt) recovery, with death, new cytotoxic therapy, relapse with marrow involvement in the absence of ANC or platelet recovery considered as competing events. Patient, disease, and CAR-T cell therapy-related variables were included in multivariable analyses. Results ANC and Plt recovery after CD19 CAR-T cell therapy were observed in 91% (ALL, 86%; CLL, 92%; NHL, 95%) and 86% (ALL, 86%; CLL, 86%; NHL, 84%) of patients, respectively. Median time to ANC recovery was 9 days and the probability of ANC recovery at day 28, 60, and 90 was 80% (95%CI, 73-87), 86% (95%CI, 80-92) and 89% (95%CI, 83-94), respectively. The probability of platelet recovery on the day of CAR-T cell infusion was 55% (95%CI, 46-64); rising to 74% (95%CI, 67-82), 83% (95%CI, 76-90), and 84% (95%CI, 77-90) on days 28, 60, and 90, respectively. A competing event was always observed in patients without ANC or Plt recovery. In multivariable analysis (Table 2), higher pre-lymphodepletion (LD) Plt count and higher peak CD8+ CAR-T cells in blood were associated with faster ANC recovery. A diagnosis of ALL was associated with slower ANC recovery. (CLL vs ALL, HR=1.60, p=0.02; NHL vs ALL, HR=2.07, p=0.007). Higher CRS grade was also associated with slower Plt recovery (HR=0.67 per grade increase, p Conclusion We observed impaired ANC and Plt recovery in a significant fraction of patients undergoing CD19 CAR-T cell therapy. Moreover, we identified factors independently associated with impaired hematopoietic recovery: disease type, prelymphodepletion Plt count, in vivo CAR-T cell expansion, and CRS grade. In addition to providing a benchmark for future CAR-T cell research, our findings suggest CRS prevention might improve hematopoietic recovery after CD19 CAR-T cell therapy.

Factors associated with durable remission after CD19 chimeric antigen receptor (CAR)-modified T-cell immunotherapy for aggressive B-cell non-Hodgkin lymphoma (NHL) have not been identified. We report multivariable analyses of factors affecting response and progression-free survival (PFS) in patients with aggressive NHL treated with cyclophosphamide and fludarabine lymphodepletion followed by 2 × 106 CD19-directed CAR T cells/kg. The best overall response rate was 51%, with 40% of patients achieving complete remission. The median PFS of patients with aggressive NHL who achieved complete remission was 20.0 months (median follow-up, 26.9 months). Multivariable analysis of clinical and treatment characteristics, serum biomarkers, and CAR T-cell manufacturing and pharmacokinetic data showed that a lower pre-lymphodepletion serum lactate dehydrogenase (LDH) level and a favorable cytokine profile, defined as serum day 0 monocyte chemoattractant protein-1 (MCP-1) and peak interleukin-7 (IL-7) concentrations above the median, were associated with better PFS. MCP-1 and IL-7 concentrations increased after lymphodepletion, and higher intensity of cyclophosphamide and fludarabine lymphodepletion was associated with higher probability of a favorable cytokine profile. PFS was superior in patients who received high-intensity lymphodepletion and achieved a favorable cytokine profile compared with those who received the same intensity of lymphodepletion without achieving a favorable cytokine profile. Even in high-risk patients with pre-lymphodepletion serum LDH levels above normal, a favorable cytokine profile after lymphodepletion was associated with a low risk of a PFS event. Strategies to augment the cytokine response to lymphodepletion could be tested in future studies of CD19 CAR T-cell immunotherapy for aggressive B-cell NHL. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

CD19-targeted chimeric antigen receptor (CAR)-modified T (CAR-T) cell immunotherapy has demonstrated impressive results in B-cell malignancies, and CAR-T cell therapies targeting other antigens are in development for other cancers. Cytokine release syndrome (CRS) and neurotoxicity can be life-threatening in a subset of patients. The severity of CRS and neurotoxicity can be impacted by the disease burden, lymphodepletion regimen, and CAR-T cell dose. Tocilizumab and corticosteroids have been used to manage these toxicities, enabling CD19 CAR-T cells to be administered without obvious compromise in efficacy. Consensus criteria for grading and managing toxicities will facilitate the widespread application of this treatment modality.

Background In relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) patients (pts) who had previously failed ibrutinib, we observed durable responses after CD19 chimeric antigen receptor-modified T-cell immunotherapy (Turtle CJ et al, JCO, 2017; NCT01865617). The combination of CD19 CAR-T cells with ibrutinib might improve response and decrease toxicity. Objectives To evaluate the efficacy and toxicity of CD19 CAR-T cell immunotherapy alone or in combination with ibrutinib for R/R CLL. Methods Our phase 1/2 study of CD19 CAR-T cell immunotherapy established a preferred regimen for R/R CLL pts of cyclophosphamide and fludarabine (Cy/Flu) lymphodepletion followed by 2 × 106 CD19 CAR-T cells/kg (Turtle, JCO, 2017). We then compared outcomes of these pts (No-ibr cohort) with a subsequent cohort receiving Cy/Flu with 2 × 106/kg JCAR014 CAR-T cells with concurrent ibrutinib (420 mg/d) from at least 2 weeks prior to leukapheresis until at least 3 months after JCAR014 infusion (Ibr cohort). Results We treated 19 and 17 pts in the No-Ibr and Ibr cohorts, respectively. Pt characteristics were comparable (Table 1). Median follow-up in responders was 764 and 98 days in the No-ibr and Ibr cohorts, respectively. Administration of ibrutinib with Cy/Flu and CD19 CAR-T cells was well tolerated in most pts; ibrutinib was reduced or discontinued in 6 pts (35%) at a median of 21 days after JCAR014 infusion. In the Ibr cohort 1 pt with grade 2 CRS (Lee, Blood, 2014) developed fatal presumed cardiac arrhythmia and 1 pt developed a subdural hematoma in the setting of trauma and thrombocytopenia. Although the proportions of pts with grade ≥1 CRS were similar between cohorts (76% vs 89%, P = 0.39), the severity of CRS (grade ≥3 CRS: Ibr, 0%; No-Ibr, 26%; P = 0.05) and serum peak IL-8 (P = 0.04), IL-15 (P = 0.003) and MCP-1 (P = 0.004) concentrations were lower in the Ibr cohort. However, we found higher peak CD4+ CAR-T cell counts (P = 0.06) and comparable peak CD8+ CAR-T cell counts (P = 0.29) in blood in the Ibr compared to the No-Ibr cohort. Sixteen pts (94%) and 18 pts (95%) in the Ibr and No-ibr cohorts, respectively, were evaluable for response. We observed a higher proportion of responders (complete response and partial response) by 2008 IWCLL criteria in the Ibr compared to the No-ibr cohort (88% vs 56%, respectively, P = 0.06). In pts with no disease by BM flow cytometry after treatment (N = 23), a higher proportion of pts in the Ibr cohort had no malignant IgH sequences at 4 weeks (83% vs 60%, P = 0.35). Using multivariable logistic regression (Table 2), treatment in the Ibr cohort and a lower pre-treatment SUVmax were associated with a higher probability of response (Ibr cohort, OR = 14.02, P = 0.05; SUVmax, OR = 1.31 per SUV unit decrease, P Conclusion The combination of ibrutinib with CD19 CAR-T cell immunotherapy was well tolerated in most pts, and might decrease the incidence of severe CRS and improve responses in pts with R/R CLL.

Introduction CD19 chimeric antigen receptor (CAR)-T cell therapy has shown efficacy in Non-Hodgkin lymphoma (NHL), but the factors associated with durable remission have not been identified. Objectives We report multivariate analyses of factors impacting progression-free survival (PFS) in NHL patients (pts) treated with cyclophosphamide and fludarabine (Cy/Flu) lymphodepletion (LD) and CD19 CAR-T cells. Methods We conducted a phase 1/2 open-label clinical trial (NCT01865617) with the primary objective of evaluating the feasibility and safety of a defined composition of CD19 CAR-T cells after LD in pts with R/R CD19+ B-cell malignancies. PFS was defined as the time from CAR-T cell infusion until disease progression or death. Multivariate models using elastic net were performed for analysis of response and PFS. Results Characteristics of the 57 pts in the study are shown in Tbl 1. Thirty-six (63%) received high-intensity (HI) Cy/Flu and 21 (37%) received low-intensity (LI) Cy/Flu LD (Tbl 1). All pts received 2 × 106 CD19 CAR-T cells/kg. The best overall response (OR) rate was 57%, with 48% complete remission (CR). Pts who achieved CR had a median PFS not reached (median follow-up [fu] 20.2 months; mo), with only 1 relapse beyond 12 mo after CAR-T cell infusion (Fig. 1). Eight of 9 pts with indolent NHL achieved CR (89%) and none relapsed (median fu 14.5 mo). For the 47 pts with aggressive NHL, the best OR rate was 51%, with 40% CR. The median PFS of aggressive NHL pts achieving CR was 20.0 mo (median fu 26.9 mo), and 24-mo probabilities of PFS and OS were 46% and 72%, respectively. In aggressive NHL, multivariate analysis showed that the probability of CR was associated with lower pre-LD serum LDH and greater increase in serum MCP-1 concentration from LD to the day of CAR-T cell infusion. HI LD was associated with more CAR-T cells in blood, but there was no difference in response in pts who received HI (CR 14/31, 45%) compared to LI LD (CR 5/16, 31%; P = .53). Multivariate analysis in aggressive NHL showed that lower pre-LD serum LDH and higher day 0 MCP-1 and peak IL-7 after CAR-T cell infusion were associated with better PFS. The IL-7 peak after CAR-T cell infusion (median day 4) correlated with day 0 IL-7, consistent with an effect of LD. MCP-1 and IL-7 increased after LD, independent of prior bridging therapy, and their concentrations correlated with Cy/Flu LD intensity. However, LD intensity was not independently associated with CR or PFS. Independent of LD intensity, a subset of pts had a robust increase in day 0 MCP-1 and peak IL-7 after LD and achieved better responses (CR 10/16, 63%) compared to those with limited cytokine increase (CR 9/30, 30%; P = .06). Conclusion Multivariate analyses show that cytokines associated with effective LD are, unlike the intensity of Cy/Flu LD, independently associated with CR and PFS. Strategies to augment biological LD efficacy could be tested in studies of CD19 CAR-T cells for aggressive NHL.

Background CD19-targeted chimeric antigen receptor-engineered (CD19 CAR)-T cell immunotherapy has shown promising efficacy in patients with relapsed or refractory (R/R) B-cell malignancies. The potential benefits of repeat infusions of CD19 CAR-T cells are unknown, and the factors associated with response, CAR-T cell in vivo expansion, and progression-free survival (PFS) after repeat infusion of CD19 CAR-T cells have not been investigated. Methods We analyzed the outcomes of patients with R/R B-cell malignancies after a second infusion of CD19 CAR-T cells (CART2) on a phase 1/2 trial (NCT01865617) at our institution. Responses after CAR-T cell therapy were evaluated around day 28 after infusion and defined according to the 2018 NCCN guidelines for acute lymphoblastic leukemia (ALL), 2018 iwCLL for chronic lymphocytic leukemia (CLL), and the Lugano criteria for non-Hodgkin lymphoma (NHL). Logistic, Cox and linear regression were used for multivariable analyses of response, progression-free survival and peak CD8+ CAR-T in blood, respectively. Bayesian model averaging was performed for variable selection. Results Forty-four patients evaluable for response (ALL, n=14; CLL, n=11; NHL, n=19) were included in this study. The median age at the time of CART2 was 58 (range, 23-73). Patients were heavily pre-treated (median prior therapies, 6; range, 2-13), and 16 patients (36%) had bulky (≥ 5cm) nodal or extramedullary disease. The median time from the first CAR-T infusion (CART1) to CART2 was 70 days (range, 28-712). Twenty-eight patients (64%) had received a CART1 dose ≥ 2x106 CAR-T cells/kg. Fifteen patients (32%) had not responded to CART1, 22 (50%) relapsed or progressed after having initially responded (complete response [CR], n=15; partial response [PR], n=7) to CART1; 7 (16%) received CART2 in PR after CART1. All characteristics are shown in the Table. We observed responses in all disease types, including 3 of 14 ALL patients (21%; all CR/CRi), 4 of 11 CLL patients (36%; CR/CRi, n=3; partial response [PR], n=1), and 9 of 19 NHL patients (47%; CR, n=2; PR, n=7). After a median follow-up of 43 months (range, 16-66) in alive and responding patients, the estimated 4-year PFS probability in responders was 23% (95% CI, 9-59%). The 4-year overall survival probability in responders was 36% (95% CI 19-71%) compared to 24% (95% CI, 12-47) in non-responders. Multivariable logistic regression modeling identified predictors of response after CART2: CART1 lymphodepletion (high-intensity cyclophosphamide and fludarabine [CyFlu] vs no CyFlu, OR=12.19, 95% CI, 1.10-1689.85, p=0.04), and peak of in vivo CAR-T cell expansion after CART2 (OR=2.31 per log10 CD8+ CAR-T cell/µL increase, 95% CI, 1.17-5.29, p=0.01). In a multivariable Cox model, a higher peak of CD8+ CAR-T cells after CART2 (HR=0.47 per log10 CD8+ CAR-T cell/µL increase, 95%CI, 0.33-0.68, p CART1 cell dose was associated with longer PFS (HR=0.36, 95% CI, 0.16-0.86, p=0.02). This suggested that CD8+ CAR-T cell peak after CART2 and factors increasing CART2 peak (e.g. prevention of immune rejection or increase in the infused cell dose) are key elements associated with outcomes of CART2. Hence, we looked at factors associated with higher CD8+ CART2 peak. In multivariable linear regression, CART1 CyFlu predicted a higher peak of CD8+ CAR-T cells after CART2 (high-intensity CyFlu vs no CyFlu, p CyFlu being the most commonly used lymphodepletion prior to CAR-T cell therapy, we evaluated the impact of CART1 CyFlu lymphodepletion intensity by comparing high-intensity to low-intensity CyFlu in our multivariable models. Logistic regression suggested higher probabilities of response to CART2 in patients who received high-intensity compared to low-intensity CyFlu prior to CART1 (OR=3.83, 95%CI, 0.85-21.83, p=0.08). In multivariable analysis, CART1 high-intensity CyFlu was associated with higher CD8+ CAR-T cell numbers at day 60 after CART2 compared to low-intensity CyFlu (p=0.01) after adjusting for disease type and total CD19+ cell count in the blood. Conclusion Our findings suggest outcomes after second infusions of CD19 CAR-T cells might be improved with high-intensity CyFlu lymphodepletion prior to CART1 and by increasing the CAR-T cell dose at the time of CART2. Table Disclosures Hirayama: DAVA Oncology: Honoraria. Till:Mustang Bio: Patents & Royalties, Research Funding. Kiem:Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; CSL Behring: Consultancy; Magenta Therapeutics: Consultancy. Shadman:Mustang Biopharma: Research Funding; Gilead: Research Funding; Bigene: Research Funding; AstraZeneca: Consultancy; Merck: Research Funding; Atara: Consultancy; AbbVIe: Consultancy, Research Funding; Genentech, Inc.: Consultancy, Research Funding; TG Therapeutics: Research Funding; Sound Biologics: Consultancy; Pharmacyclics: Consultancy, Research Funding; ADC Therapeutics: Consultancy; Verastem: Consultancy; Celgene: Research Funding; Acerta: Research Funding; Emergent: Research Funding; Sunesis: Research Funding. Cassaday:Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Kite/Gilead: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Other: Spouse's disclosure: employment, stock and other ownership interests. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Maloney:BioLine RX, Gilead,Genentech,Novartis: Honoraria; Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; Celgene,Kite Pharma: Honoraria, Research Funding; A2 Biotherapeutics: Honoraria, Other: Stock options . Turtle:Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member; Allogene: Other: Ad hoc advisory board member; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; T-CURX: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member.

Background: Treatment of high-risk CLL remains challenging despite the introduction of novel therapeutic agents. Chimeric antigen receptor T-cell (CAR-T) therapy has shown promising efficacy in these patients (pts) (Turtle, JCO, 2017; Gauthier, ASH, 2019), but progressive disease after CAR-T is not uncommon. Understanding the outcomes of pts with relapsed or refractory CLL after CAR-T is important for establishing a benchmark for trials in this setting and to study optimal treatment sequencing in high-risk CLL. Methods: CLL pts treated with CD19-specific CAR-T on a clinical trial (NCT01865617) at Fred Hutch/University of Washington were reviewed. Using the 2018 IWCLL definitions, we identified pts who had stable or progressive disease (SD/PD) on initial assessment (4 weeks) or had a relapse after a complete or partial response (CR/PR) at any time. Overall survival (OS) was calculated using the Kaplan-Maier estimates. For univariate models, we included del17p, complex karyotype (CK), ibrutinib (IB) and venetoclax (Ven) failure before CAR-T, number of prior treatment lines, Richter's transformation (RT), use of bridging treatment, bulky disease (>5cm) before CAR-T, cytokine release syndrome (CRS; ≥ grade 3), neurotoxicity (NT; ≥ grade 3), refractory vs. relapsed (after CAR-T), use of IB, Ven, idelalisib (Idela), repeat CAR-T or allogeneic transplant (allo-SCT) after CAR-T progression. Only factors with significant association in the univariable models (p < 0.05) were included in the multivariable analysis. Results: 28 pts were identified with refractory (n=16; 57%) or relapsed disease [n=12; 43% - median time to relapse after CAR-T was 11 months (1.8-23)] after CAR-T. Median age was 60 (41-70) and 25% were female. Nine pts (32%) had a history of RT. Cytogenetic changes were: del17p (71%), CK (78%), del11q (37%), del13q (43%) and +12 (11%). Details of pre-CAR-T treatments are outlined in table-1. Before CAR-T, 24 pts (86%) had progressed on IB, 10 (36%) on Ven and 6 (21%) on Idela. Nine pts (32%) had progressed on both IB and Ven before CAR-T and 5 pts (18%) had prior allo-SCT. Five pts (18%) received bridging therapy after leukapheresis. Lymphodepletion (LD) was with cyclophosphamide and fludarabine (CyFlu) in 25 pts (89%). CAR-T adverse events included CRS in 22 pts (grade 3 in 2 pts, no grade 4) and NT in 7 (grade 3 in 5 and no grade 4). Details of Post CAR-T failure treatments are summarized in table-2 Both IB and Ven were used after CAR-T even if pts had progressed on them before. For Ven, 5 of 11 patients who received Ven after CAR-T had progressed on it before but had a median duration of response (DOR) of 5 months (2-8) which was not different from pts who were Ven responsive before CAR-T [5 months (1-10); p = 0.95). This was not true for IB with DOR of 2 months (0.5-7) in pre-CAR-T IB failed vs. 12.25 months (6-18.5) in pre CAR-T IB responsive pts (p = 0.001). Fourteen pts (51%) had repeat CAR-T therapy on the same protocol and 6 had CR (3) or PR (3). Four of these patients relapsed later with a DOR after second CAR-T of 5.5 months (1-33). Six pts (23%) received an allo-SCT median 6.5 months after CAR-T progression (2-14) and 3 achieved a CR (2) or PR (1), 2 did not respond (PD=1; SD=1) and one died before assessment. Two of these pts relapsed 9 and 39 months after allo-SCT. Chemotherapy was used in 7 pts with no responses. From variables tested in univariable models, only 4 were entered to the multivariate analysis (p < 0.05) and all remained significantly associated with OS: history of progression on both IB and Ven before CAR-T was strongly associated with poor OS [hazard ratio (HR) 11.2 (95% CI: 2.5-50.5) ; p=0.002]. Receiving an allo-SCT after CAR-T progression was associated with an improved OS [HR 0.14 (95% CI: 0.02-0.97); p= 0.04]. Other factors were post CAR-T progression treatment with IB [HR 0.16 (95% CI: 0.04-0.68); p = 0.01] or Ven [HR 0.17 (95% CI: 0.03-0.8); p = 0.02]. Median OS for the entire cohort was 10.4 months (0.23-59). [Fig-1] Conclusion: This data sets a benchmark for clinical trials that intend to improve outcome of CLL pts with progression after CAR-T. OS after CAR-T progression was significantly shorter in pts who received CAR-T after failing both IB and Ven compared to others. This finding supports referring high-risk CLL pts for CAR-T treatment while still responsive to either IB (BTKis) or Ven. For eligible pts, allo-SCT seems to provide a higher chance of survival in pts who progress after CAR-T. Disclosures Shadman: Verastem: Consultancy; Acerta Pharma: Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutic: Research Funding; Pharmacyclics: Consultancy, Research Funding; Sound Biologics: Consultancy; AbbVie: Consultancy, Research Funding; ADC Therapeutics: Consultancy; Gilead: Consultancy, Research Funding; Celgene: Research Funding; Mustang Bio: Research Funding; Sunesis: Research Funding; BeiGene: Research Funding; Atara Biotherapeutics: Consultancy; Astra Zeneca: Consultancy. Hirayama:DAVA Oncology: Honoraria. Lynch:Takeda Pharmaceuticals: Research Funding; Juno Therapeutics: Research Funding; Rhizen Pharmaceuticals S.A: Research Funding; T.G. Therapeutics: Research Funding; Incyte Corporation: Research Funding; Johnson Graffe Keay Moniz & Wick LLP: Consultancy. Smith:Pharmacyclics: Research Funding; Portola Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme Corp: Consultancy, Research Funding; Bristol-Myers Squibb (spouse): Research Funding; Denovo Biopharma: Research Funding; Ayala (spouse): Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Ignyta (spouse): Research Funding; Acerta Pharma BV: Research Funding. Ujjani:AbbVie: Honoraria, Research Funding; Pharmacyclics: Honoraria; PCYC: Research Funding; Astrazeneca: Consultancy; Genentech: Honoraria; Gilead: Consultancy; Pharmacyclics: Honoraria; PCYC: Research Funding; Atara: Consultancy. Kiem:Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; CSL Behring: Consultancy; Magenta Therapeutics: Consultancy. Till:Mustang Bio: Patents & Royalties, Research Funding. Gopal:Teva, Bristol-Myers Squibb, Merck, Takeda, Seattle Genetics, Pfizer, Janssen, Takeda, and Effector: Research Funding; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte.: Consultancy; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte: Honoraria. Maloney:Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; Celgene,Kite Pharma: Honoraria, Research Funding; BioLine RX, Gilead,Genentech,Novartis: Honoraria; A2 Biotherapeutics: Honoraria, Other: Stock options . Turtle:Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Humanigen: Other: Ad hoc advisory board member; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Allogene: Other: Ad hoc advisory board member; Kite/Gilead: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member. OffLabel Disclosure: CAR-T is not an approved treatment for CLL

Introduction Autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) have produced impressive minimal residual disease-negative complete remission (MRD-neg CR) rates in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) patients (pts). Factors associated with durable remission in pts achieving MRD-neg CR after immunotherapy with T cells engineered with a CD19 CAR (FMC63.41bb.3ζ) have not been elucidated. Methods We studied factors impacting disease-free survival (DFS) of adults with B-ALL treated with lymphodepletion chemotherapy and CD19 CAR-T cells in a phase I/II clinical trial (NCT01865617). Pts were eligible for this analysis if they had bone marrow leukemia identified by flow cytometry and/or extramedullary disease before CAR-T cell therapy, and received CAR-T cells at or below the previously determined maximum tolerated dose (MTD; Turtle, JCI 2016). Anti-tumor response after CAR-T cell infusion was assessed by bone marrow aspiration and biopsy, with PET-CT performed in pts with extramedullary disease. High resolution (1:10,000) flow cytometry was used to identify marrow MRD, and marrow from pts in MRD-neg CR was evaluated by high throughput sequencing (HTS) of IGH, IGK, TRB, TRD, and TRG genes. Cox regression univariate and stepwise multivariable modeling were performed to identify factors associated with DFS. Results Of 57 pts who received lymphodepletion and CD19 CAR-T cells, 53 were evaluable for response and 4 were not evaluable (one MRD-neg CR before CAR-T cell infusion; 2 received CAR-T cells above the MTD; one fatal neurotoxicity prior to restaging). Forty-five of 53 restaged pts (85%) achieved MRD-neg CR after CAR-T cell therapy. With a median follow-up of 30.9 months, DFS and overall survival (OS) were longer in pts who achieved MRD-neg CR compared to those who did not (Fig 1A; median DFS, 7.6 vs 0.8 months, P < .0001; median OS, 20.0 vs 5.0 months, P = .014). Twenty-eight of the 45 pts who achieved MRD-neg CR had a leukemic clone identified by HTS prior to CAR-T cell infusion, and in 20 of these pts (71%), the leukemic clone was not detected in marrow 3 weeks after CAR-T cell infusion. DFS was better in MRD-neg CR pts with no detected malignant clone compared to those with a persistent clone by HTS (Fig 1B; median DFS, 8.4 vs 3.6 months, P = .036). We then used stepwise multivariable modeling to determine factors impacting DFS in the pts who achieved MRD-neg CR (n = 45). Better DFS was seen in pts with a higher pre-lymphodepletion platelet count (hazard ratio, HR 0.65 [95% CI; 0.47-0.88] per 50,000/μL increment P = .006), lower pre-lymphodepletion LDH (HR 1.39 [1.12-1.74] per 100 U/L increment, P = .003), and with incorporation of fludarabine into the cyclophosphamide-based lymphodepletion (Cy/Flu; HR 0.34 [0.15-0.78], P = .011). Similar findings were noted in analysis of MRD-neg CR pts who had no malignant clone by HTS after CAR-T cells. Pts with platelets ≥100,000/μL and normal LDH before lymphodepletion who received Cy/Flu (good risk, n = 15) had 2-year point estimates of DFS and OS of 78% and 86%, respectively. Allogeneic hematopoietic cell transplantation (HCT) is standard of care in suitable R/R adult B-ALL pts after achieving MRD-neg CR. Eighteen pts in MRD-neg CR after CAR-T cells underwent HCT a median of 2.3 months after CAR-T cell infusion. We analyzed the effect on DFS of HCT after CAR-T cell therapy by treating HCT as a time-dependent covariate. After adjusting for LDH, platelets, and Cy/Flu lymphodepletion, pts undergoing HCT after CAR-T cell therapy had lower risk of failure for DFS compared to those who did not undergo HCT (Table 1). No significant interaction effect was seen between HCT and risk group (P = .51). With a median follow-up of 28.4 months after HCT, 2-year point estimates of DFS and OS were 61% and 72% respectively. The 2-year cumulative incidence of relapse was 17% and non-relapse mortality was 23%. Conclusion A high rate of MRD-neg CR was seen following CD19 CAR-T cell therapy in adult B-ALL pts and is associated with improved DFS and OS. Absence of the index clone by HTS after CAR-T cells was associated with better DFS, suggesting deeper responses are associated with improved outcomes. Stepwise multivariable modeling identifies better DFS in pts with higher pre-lymphodepletion platelet count and lower LDH, and with use of Cy/Flu lymphodepletion. After adjusting for these factors, HCT after CD19 CAR-T cells may also be associated with better DFS. Disclosures Hay: DAVA Oncology: Honoraria. Hirayama:DAVA Oncology: Honoraria. Li:Juno Therapeutics: Employment, Equity Ownership. Till:Mustang Bio: Patents & Royalties, Research Funding. Kiem:Rocket Pharmaceuticals: Consultancy; Homology Medicine: Consultancy; Magenta: Consultancy. Ramos:Seattle Genetics: Employment, Equity Ownership. Shadman:Qilu Puget Sound Biotherapeutics: Consultancy; AstraZeneca: Consultancy; Pharmacyclics: Research Funding; Genentech: Consultancy; Mustang Biopharma: Research Funding; AbbVie: Consultancy; Celgene: Research Funding; Verastem: Consultancy; Acerta Pharma: Research Funding; Genentech: Research Funding; TG Therapeutics: Research Funding; Beigene: Research Funding; Gilead Sciences: Research Funding. Cassaday:Adaptive Biotechnologies: Consultancy; Amgen: Consultancy, Research Funding; Incyte: Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Research Funding; Pfizer: Consultancy, Research Funding; Merck: Research Funding. Acharya:Juno Therapeutics: Research Funding; Teva: Honoraria. Riddell:Adaptive Biotechnologies: Consultancy; NOHLA: Consultancy; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees. Maloney:Seattle Genetics: Honoraria; Roche/Genentech: Honoraria; GlaxoSmithKline: Research Funding; Janssen Scientific Affairs: Honoraria; Juno Therapeutics: Research Funding. Turtle:Adaptive Biotechnologies: Consultancy; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Gilead: Consultancy; Nektar Therapeutics: Consultancy, Research Funding; Caribou Biosciences: Consultancy; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy; Aptevo: Consultancy.