CD19 Chimeric Antigen Receptor T-cell Research Articles

An assessment model for the efficacy of autologous CD19 chimeric antigen receptor T-cell therapy and relapse or refractory diffuse large B-cell lymphoma risk.

An assessment model for the efficacy of autologous CD19 chimeric antigen receptor T-cell therapy and relapse or refractory diffuse large B-cell lymphoma risk.

Five-Year Follow-Up of Standard-of-Care Axicabtagene Ciloleucel for Large B-Cell Lymphoma: Results From the US Lymphoma CAR T Consortium.

Axicabtagene ciloleucel (axi-cel) is an autologous CD19 chimeric antigen receptor (CAR) T-cell therapy that is approved for the treatment of relapsed or refractory large B-cell lymphoma. Little is known about the long-term survivorship after CAR T-cell therapy. We previously reported the results of 298 patients who were leukapheresed with the intent to receive standard-of-care axi-cel (n = 275 infused) after two or more previous lines of therapy at a median follow-up of 12.9 months. Here, we report extended follow-up of this cohort to a median of 58 months, with a focus on late survivorship events. Among axi-cel-infused patients, progression-free survival at 5 years was 29% and overall survival (OS) at 5 years was 40%. The 5-year lymphoma-specific survival was 53% with infrequent late relapses. However, the 5-year nonrelapse mortality (NRM) was 16.2%, with over half of NRM events occurring beyond 2 years. Patients who were 60 years and older had a lower risk of relapse (P = .02), but a higher risk of NRM compared with patients younger than 60 years (NRM odds ratio, 4.5 [95% CI, 2.1 to 10.8]; P < .001). Late NRM was mainly due to infections and subsequent malignant neoplasms (SMNs). In total, SMNs occurred in 24 patients (9%), including therapy-related myeloid neoplasms (n = 15), solid tumors (n = 7), and unrelated lymphoid malignancies (n = 2). In the standard-of-care setting, axi-cel exhibits outcomes consistent with those reported in clinical trials, with sustained, durable responses observed at the 5-year time point. However, late infections and the development of SMN are key survivorship issues that reduce long-term survival after CAR T-cell therapy, particularly in the elderly.

Aggressive Lymphoma after CD19 CAR T-Cell Therapy

SummaryThe development of a fatal, clonal, autonomously proliferating CD4−CD8− chimeric antigen receptor (CAR)+ peripheral T-cell lymphoma (PTCL) occurred 1 month after a patient received treatment with tisagenlecleucel for relapsed primary central nervous system lymphoma. The PTCL had a clonal T-cell receptor rearrangement, which was already detectable in the apheresis product for CAR T-cell manufacturing and 7 months earlier for autologous transplantation. Somatic DNMT3A and TET2 mutations in CD34+ stem cells and their progeny were detected in the PTCL, in the apheresis specimen that was obtained for CAR T-cell production, and in the autotransplant. The PTCL harbored an additional somatic TET2 mutation, which was already detectable in the CAR T-cell apheresis product and the final CAR T-cell product at very low frequencies, providing evidence that clonal hematopoiesis had contributed to lymphomagenesis.

Shift from Widespread to Tailored Antifungal Prophylaxis in Lymphoma Patients Treated with CD19 CAR T Cell Therapy: Results from a Large Retrospective Cohort

BackgroundPatients undergoing CD19 chimeric antigen receptor (CAR) T-cell therapy exhibit multiple immune deficits that may increase their susceptibility to infections. Invasive fungal infections (IFI) are life-threatening events in the setting of hematological diseases. However, there is ongoing debate regarding the optimal role and duration of antifungal prophylaxis in this specific patient population. ObjectivesThe objective of this study was to provide a comprehensive overview of the evolution of IFI prophylactic strategies over time and to assess IFI incidence rates in a cohort of patients with relapsed or refractory (R/R) lymphoma treated with CAR-T cell therapy. Study DesignSingle-center, retrospective study from a cohort of patients with R/R B-cell lymphoma treated with CD19 CAR-T cell therapy between April 2016 and March 2023. Group A (April 2016- August 2020) consisted of patients primarily treated with fluconazole, irrespective of their individual IFI risk profile. In Group B (September 2020- March 2023) antifungal prophylaxis was recommended only for high-risk patients. ResultsOverall, 330 patients were included. Antifungal prophylaxis was prescribed to 119/142 (84%) patients in Group A and 58/188 (31%) in Group B (p<0.001). Anti-mold azoles were prescribed to 8 (5.6%) patients in Group A and 21 (11.2%) patients in Group B. In Group A, 42 (29%) patients switched to another antifungal, 9 (21%) because of toxicity, with 6 cases of transaminitis and 3 cases of prolonged QTc. In Group B, 21 (11.2%) patients switched the antifungal drug, mainly from fluconazole or micafungin to a mold-active agent following revised guidelines. No difference was found in liver toxicity between the two groups at infusion, day 10, and day 30. No significant differences were observed between the groups. IFI following CAR T-cell therapy were rare, with one case of cryptococcal meningoencephalitis in group A (0.7%) and one case of invasive aspergillosis in group B (0.5%), both occurring in patients on micafungin prophylaxis. ConclusionIn this large single-center cohort of patients with R/R lymphoma treated with CAR T-cells, we show that individualized prophylaxis, alongside careful management of CAR T-cell related toxicities like CRS, was associated with a very low IFI rate, avoiding the risk of unnecessary toxicities, drug-drug interactions, and high costs.

Comprehensive characterization of cytopenia after chimeric antigen receptor-T cell infusion in patients with relapsed or refractory multiple myeloma

BackgroundMany studies have demonstrated the effectiveness of chimeric antigen receptor-T (CAR-T) cell therapy for relapsed or refractory multiple myeloma (RRMM), but the hematologic toxicity has not been well characterized. MethodsA total of 111 adults with RRMM who received BCMA CAR-T cells, BCMA + CD19 CAR-T cells or tandem BCMA/CD19 dual-target (BC19) CAR-T cells infusion were enrolled. We characterized cytopenia and hematologic recovery at different time points after CAR-T-cell therapy, analyzed the effect of cytopenia on prognosis and identified the risk factors. ResultsPatients had a high probability of cytopenia, with anemia, neutropenia and thrombocytopenia occurring in 92%, 95% and 73%, respectively. There were 60 (54%) patients had prolonged hematologic toxicity (PHT) after D28. The median hemoglobin and platelet count were significantly lower at D28 post-CAR-T cell therapy than at baseline. Hemoglobin increased to above baseline at D90. The median absolute neutrophil count was lower than baseline at D0 and D28, and it recovered to baseline at D180. The baseline level of lactate dehydrogenase was associated with thrombocytopenia. Extramedullary involvement was associated with hemoglobin recovery, while the baseline level of albumin and types of CAR-T were related to platelet recovery. Patients with anemia at baseline and at D0, D180 and D360 had shorter progression-free survival (PFS), while anemia at D0, D60, D180 and D360 was associated with shorter overall survival (OS). Neutropenia at D0 was associated with shorter PFS and patients with neutropenia at D90 or D180 had shorter OS. Patients with thrombocytopenia at any time had shorter PFS and OS. Compared to patients without PHT, patients with PHT had shorter PFS and OS. ConclusionsThe majority of RRMM patients treated with CAR-T cells experienced cytopenia. Cytopenia occurred at specific time points was associated with a poorer prognosis.

CD19 CAR-T treatment shows limited efficacy in r/r DLBCL with double expression and TP53 alterations

ObjectAutologous CD19 chimeric antigen receptor T-cell therapy (CAR-T) significantly modifies the natural course of chemorefractory diffuse large B-cell lymphoma (DLBCL). However, 25% to 50% of patients with relapsed/refractory DLBCL still do not achieve remission. Therefore, investigating new molecular prognostic indicators that affect the effectiveness of CAR-T for DLBCL and developing novel combination therapies are crucial. MethodsData from 73 DLBCL patients who received CD19 CAR-T (Axi-cel or Relma-cel) were retrospectively collected from Shanghai Tongji Hospital of Tongji University, The Second Affiliated Hospital Zhejiang University School of Medicine, and The Affiliated People's Hospital of Ningbo University. Prior to CD19 CAR-T-cell transfusions, the patients received fludarabine and cyclophosphamide chemotherapy regimen. ResultsOur study revealed that relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) patients with both Double-expression (MYC > 40% and BCL2 > 50%) and TP53 alterations tend to have a poorer clinical prognosis after CAR-T therapy, even when CAR-T therapy is used in combination with other therapies. However, CAR-T therapy was found to be effective in patients with only TP53 alterations or DE status, suggesting that their prognosis is in line with that of patients without TP53 alterations or DE status. ConclusionsOur study suggests that r/r DLBCL patients with both DE status and TP53 alterations treated with CAR-T therapy are more likely to have a poorer clinical prognosis. However, CAR-T therapy has the potential to improve the prognosis of patients with only TP53 alterations or DE status to be similar to that of patients without these abnormalities.

CD19 chimeric antigen receptor-T cells as bridging therapy to allogeneic hematopoietic cell transplantation improves outcome in patients with refractory/relapsed B-cell acute lymphoblastic leukemia

Chimeric antigen receptor (CAR)-T cell therapy has been confirmed improving remission rates in refractory patients or relapsed B-cell acute lymphoblastic leukemia (R/R B-ALL). However, the added benefits of undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) following CAR-T therapy remain a subject of debate. In this research we investigated the efficiency and long-term outcomes of CD19 CAR-T bridging with allo-HSCT in R/R B-ALL patients. A total of 42 patients were brought into the cohort studies. Our findings revealed that patients who appected CAR-T followed by HSCT had a 1-year overall survival (OS) rate of 70 % and a 1-year leukemia-free survival (LFS) rate of 95 %. Moreover, patients who underwent this combined treatment had higher OS and LFS rates compared to those who received CAR-T therapy alone. In conclusion, the results of this clinical trial provide compelling evidence for the safety and efficacy of using CAR-T therapy as a bridging strategy to allo-HSCT in patients with R/R B-ALL.

Efficacy and safety of bendamustine-containing bridging therapy in R/R LBCL patients receiving CD19 CAR T-cells.

Bridging therapy (BT) after leukapheresis is required in most relapsed/refractory (R/R) large B-cell lymphoma (LBCL) patients receiving chimeric antigen receptor (CAR) T cells. Bendamustine-containing regimens are a potential BT option. We aimed to assess if this agent had a negative impact on CAR-T outcomes when it was administered as BT. We included R/R LBCL patients from six centers who received systemic BT after leukapheresis from February 2019 to September 2022; patients who only received steroids or had pre-apheresis bendamustine exposure were excluded. Patients were dividedinto two BT groups, with and without bendamustine. Separate safety and efficacy analyses were carried out for axi-cel and tisa-cel. Of 243 patients who received BT, bendamustine (benda) was included in 62 (26%). There was a higher rate of BT progressors in the non-benda group (62% vs. 45%, p = 0.02). Concerning CAR-T efficacy, complete responses were comparable for benda versus non-benda BT cohorts with axi-cel (70% vs. 53%, p = 0.12) and tisa-cel (44% vs. 36%, p = 0.70). Also, 12-month progression-free and overall survival were not significantly different between BT groups with axi-cel (56% vs. 43% and 71% vs. 63%) and tisa-cel (25% vs. 26% and 52% vs. 48%); there were no differences when BT response was considered. CAR T-cell expansion for each construct was similar between BT groups. Regarding safety, CRS G ≥3 (6% vs. 6%, p = 0.79), ICANS G ≥3 (15% vs. 17%, p = 0.68), severe infections, and neutropenia post-infusion were comparable among BT regimens. BT with bendamustine-containing regimens is safe for patients requiring disease control during CAR T-cell manufacturing.

Real-world barriers to CAR-T access: A Canadian referral center perspective.

e19002 Background: CD19 chimeric antigen receptor T-cell (CAR-T) is the standard of care for patients with relapsed or refractory large B-cell lymphoma (LBCL) beyond second line therapy. Access to CAR-T remains a significant barrier for patients and is limited by the numbers of CAR-T centers, patient comorbidities and lymphoma kinetics. This study aims to define clinical features associated with CAR-T ineligibility and associated outcomes in a real-world setting. Methods: We conducted a single center retrospective study of all adult patients with R/R LBCL referred at Hôpital Maisonneuve-Rosemont in Montreal, Canada, for CAR-T between July 2019 and December 2023. Comprehensive data were gathered from electronic medical records. This study was approved by the institutional ethics committee. Results: A total of 235 patients were evaluated: 133 (57%) were infused, 10 (4%) were apheresed but not infused and 92 (39%) patients were deemed ineligible and further analyzed. The main reasons for ineligibility were ECOG performance score more than 1 (n=23), comorbidities (n=18), not R/R to two prior lines of therapy (LOT) (n=15) and rapid disease progression (n=15). Prohibitive comorbidities included: creatinine clearance &lt;45ml/min (n=13), left ventricular ejection fraction &lt;45% (n=2), unstable coronary artery disease (n=1), severe valvular disease (n=1) and Child B cirrhosis (n=1). 10 declined CAR-T and 10 were diagnosed or had lymphoma subtypes not covered by CAR-T reimbursement criteria: grade 3B follicular lymphoma (n=5), T-cell lymphoma (n=2), Hodgkin lymphoma (n=1), Richter from chronic lymphocytic leukemia (n=1) and transformed marginal zone lymphoma (n=1). 33 had more than one exclusion criteria. Median age at referral was 66 years (range: 25-87). Median IPI at diagnosis (available in 74%) was 3 (range: 0-5). At CAR-T evaluation, 77 patients had a stage 3 or 4 disease and 7 had CNS involvement. Median LOT for LBCL were 2 (range: 0-5), 46 were primary refractory and 23 relapsed within 12 months. 18 patients had undergone prior high dose therapy and autologous stem cell transplant (HDT-ASCT). 6 were exposed to novel therapies (polatuzumab=6, mosunetuzumab=2). Median follow-up after CAR-T exclusion was 4.25 months (range: 0-49.2). 50 (68% of patients with B-cell lymphoma R/R to at least 2 LOT) were deceased at time of analysis. Data on further LOT was available for 22 patients: 10 received novel therapies (tafasitamab=5, polatuzumab=3, mosunetuzumab=1, alectinib=1), 6 HDT-ASCT, 3 allogeneic stem cell transplant, 4 conventional chemotherapies and 2 radiation therapy. Conclusions: 39% of evaluated patients with R/R LBCL were ineligible for CAR-T mainly due to poor performance status, comorbidities and prohibitive progressive disease. Our study did not capture patients not referred due to geographical considerations but highlights dismal outcomes for patients ineligible for CAR-T. Widespread access to novel therapies is awaited.

Factors associated with manufacturing failure of commercial CD19 CAR-T cell products for large b cell lymphoma (LBCL).

7044 Background: As CD19 chimeric antigen receptor T-cell (CAR-T) therapy has shown curative potential in patients (pts) with relapsed/refractory (R/R) LBCL, indications for treatment are expanding. The reported manufacturing failure rate was 1-13% in clinical trials, but there is poor understanding of out-of-specification (OOS) products and CAR-T products on 2nd apheresis. This study aims to characterize factors associated with successful manufacturing. Methods: This study includes pts with R/R LBCL who underwent apheresis for CAR-T product between 12/2017 and 2/2023. Baseline variables were obtained at time of apheresis. OOS product is defined as not meeting eligibility for a commercial product but suitable for infusion on expanded access protocol. 2nd apheresis was performed due to initial manufacture failure. Progression-free survival (PFS) is the time from CAR-T conditioning to disease progression or death, whichever occurs first. Overall survival (OS) is defined as time from conditioning to death or last follow-up. A chi-square test or Fisher’s exact test was used for comparison of 2 categorical variables. Results: 319 pts underwent apheresis; 261(78%) were axicabtagene ciloleucel, 26(8%) were tisagenlecleucel and 47(14%) were lisocabtagene maraleucel 284(85%); products were in-specification (in-spec) on 1st apheresis, 8(2%) in-spec on 2nd apheresis, 27(8%) OOS, and 15(4%) manufacturing failures. Comparison of variables are in the table. OOS rates were 10/250(4%) for axicabtagene, 4/23(17.4%) for tisagenlecleucel and 13/46(28.3%) for lisocabtagene. Reason for OOS product were low viability:14, high interferon:4, internal manufacturing issue:2, CD4 lineage purity:2, high transduction:1, low transduction:2, and low vector copies:2. No association was found with clinical factors and OOS. Factors associated with OOS were advanced stage (P=0.012), IPI score &gt;3 (p=0.001), extranodal involvement (P=0.012) and older age (p= 0.002). In-spec product on 1st apheresis had greater OS (p= 0.011) and PFS (p=0.043) than 2nd apheresis. Conclusions: Factors predictive of OOS product include older age, advanced disease, extranodal involvement and high IPI. Pts requiring 2nd apheresis had worse survival. Lisocabtagene had higher likelihood for OOS product. Outcomes for OOS products are not well described. Failure to manufacture an eligible product is a barrier for LBCL pts to receive CAR-T therapy. [Table: see text]

Impact of late immune effector cell associated hematotoxicity (ICAHT) and prolonged neutropenia after CAR-T therapy on infection and survival outcomes.

7041 Background: Prolonged hematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CART) therapy. The EHA/EBMT panel developed the late ICAHT grading which incorporates depth of neutropenia after Day 30. Recently, the early ICAHT grade (D 0-30) was associated with infection (infx) and survival outcomes. Here, we evaluate the impact of late ICAHT and neutropenia on survival and infx outcomes after CD19 CART. Methods: We included two cohorts of adult patients (pts) with large B cell lymphoma (LBCL), mantle cell lymphoma (MCL), and follicular lymphoma (FL) who received commercial CD19 CART. Late was graded based on depth of neutropenia after D30 (G1 ≤ 1.5; G2 ≤ 1.0; G3 ≤ 0.5; G4 ≤ 0.1 G/L) and pts were censored for relapse, progression, or loss of follow up. Infxs after D30 were graded per CTCAE as mild G ≤ 2 or severe G ≥ 3. Cohort 1 included pts from 8 US medical centers for survival outcomes. Only data for severe infx was collected from D30-D100 and longer term infx data was not available. Landmark survival analysis at D+100 was performed using the Kaplan-Meier method and multivariate cox proportional hazards. To assess long term late infx outcomes, cohort 2 included pts at Vanderbilt with granular cytopenia and infx data (D30-365). Results: Cohort 1 included 277 pts with LBCL of whom 143 received axi-cel, 61 tisa-cel, and 72 liso-cel with median follow up 395 days (range 30-1763). By D100, there were 111 (40%) without, 89 (41%) with mild to moderate (G1-G2), and 53 (19%) with severe (G3-G4) late ICAHT. There were 13 pts (6%) with severe infx which was not associated with severe ICAHT (p=0.68). In multivariate landmark survival analysis using previously published covariates, severe late ICAHT was not associated with PFS (HR 1.64, p=0.11) or OS (HR 1.54, p=0.17); however, D100 neutropenia (ANC ≤ 1000) was associated with inferior PFS (HR 2.00, p=0.002) and OS (HR 1.95, p=0.003). NRM was 11% and associated with D100 neutropenia (p=0.015) but not late ICAHT (p=0.15). Cohort 2 had 43 pts (35 LBCL, 7 MCL, 1 FL) with median follow up 483 days (range 233-2133). 34 pts received axi-cel, 2 tisa-cel, and 7 brexu-cel. By 1 yr, there were 10 (23%) without, 27 (63%) with mild to moderate, and 6 (14%) with severe late ICAHT. There were 52 infxs by 1 yr and 16 pts (37%) had no infx. Median time to first infx after D30 was 165 days (range 31-365). There were 31 (60%) mild and 21 (40%) severe infx. IVIG was used in 28 (65%) and G-CSF in 27 (62%). Only 1 pt with severe ICAHT had severe infx. Late ICAHT and neutropenia were not associated with severe infection in logistic regression. Conclusions: These data did not show an association between EHA/EBMT late ICAHT grading and infx after D30 or survival. A detailed reporting of T and B cell reconstitution data may provide clues to better predict late infx. Persistent neutropenia at D100 was associated with inferior PFS and OS which needs validation in future studies.

CD19 CAR T cells for multiple sclerosis: Forging further into the new frontier

The efficacy of CD19 chimeric antigen receptor (CAR) Tcells in B cell malignancies has generated recent interest in their application to other B cell-related pathologies, such as autoimmune diseases. Fischbach etal.1 report on the use of CD19 CAR Tcells in two patients with progressive multiple sclerosis, demonstrating feasibility and safety for the first time in this disease process.

Emerging Innate Immune Cells in Cancer Immunotherapy: Promises and Challenges.

Immune checkpoint inhibitor (ICI)-based therapy has made an unprecedented impact on survival benefit for a subset of cancer patients; however, only a subset of cancer patients is benefiting from ICI therapy if all cancer types are considered. With the advanced understanding of interactions of immune effector cell types and tumors, cell-based therapies are emerging as alternatives to patients who could not benefit from ICI therapy. Pioneering work of chimeric antigen receptor T (CAR-T) therapy for hematological malignancies has brought encouragement to a broad range of development for cellular-based cancer immunotherapy, both innate immune cell-based therapies and T-cell-based therapies. Innate immune cells are important cell types due to their rapid response, versatile function, superior safety profiles being demonstrated in early clinical development, and being able to utilize multiple allogeneic cell sources. Efforts on engineering innate immune cells and exploring their therapeutic potential are rapidly emerging. Some of the therapies, such as CD19 CAR natural killer (CAR-NK) cell-based therapy, have demonstrated comparable early efficacy with CD19 CAR-T cells. These studies underscore the significance of developing innate immune cells for cancer therapy. In this review, we focus on the current development of emerging NK cells, γδ T cells, and macrophages. We also present our views on potential challenges and perspectives to overcome these challenges.

Impact of race and social determinants of health on outcomes in patients with aggressive B-cell NHL treated with CAR-T therapy

AbstractChimeric antigen receptor (CAR) T-cell (CAR-T) immunotherapy is an effective therapy for relapsed/refractory B-cell non-Hodgkin lymphoma (r/r B-NHL). However, data are limited on the impact of the convergence of race and social determinants of health on outcomes for patients treated with CAR-T therapy. We examined the impact of interactions between race and insurance type on health care use and outcomes in patients treated with CAR-T therapy for aggressive B-NHL. Adult patients with r/r B-NHL treated with CD19 CAR-Ts were identified between 2015 and 2021 across 13 US academic centers. Insurance type, demographic, and clinical data were collected and analyzed. In total, 466 adult patients were included in our analysis. Median follow-up after CAR-T therapy was 12.7 months. Median progression-free survival (mPFS) was longer for Caucasians (11.5 months) than for African Americans (3.5 months; hazard ratio [HR], 1.56 [1.03-2.4]; P = .04) or Asians (2.7 months; HR, 1.7 [1.02-2.67]; P = .04). Differences in median overall survival (mOS) were not significant. For Medicare (n = 206) vs Medicaid (n = 33) vs private insurance (n = 219) vs self-pay (n = 7): mPFS was 15.9 vs 4.2 vs 6.0 vs 0.9 months (P < .001), respectively; and mOS was 31.2 vs 12.8 vs 21.5 vs 3.2 months (P < .001), respectively. Our multicenter retrospective analysis showed that race and insurance status can affect outcomes for patients treated with CAR-T therapy.

Abstract 4002: Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as a promising approach for treating hematological malignancies, particularly B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to delve into the activation dynamics and therapeutic efficacy of CD19 CAR-T cells in a disseminated NALM6-luc human B-ALL murine model. Experimental design: NALM6-Luc-mCh-Puro cells were implanted intravenously in female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Animals were treated with CD19 CAR-T cells alone and in combination with ibrutinib. The anti-tumor activity was assessed, and the persistence and activation of CAR-T cells in the blood were evaluated using flow cytometry. Additionally, ddPCR analysis was employed to quantify CAR copy numbers, providing insights into the T-cell persistence over time. Results: Treatment with CD19 CAR-T cells exhibited notable anti-cancer activity and that activity was enhanced in combination with ibrutinib, in line with other reports. The increased time to progression was noted as 69.2% for the CAR-T group and 138.4% for the combination of CAR-T and ibrutinib compared to control. Flow cytometry analysis showed that CAR-T cells persisted in the blood throughout the study. The mean CAR-T absolute cell count for both CAR-T and CAR-T plus ibrutinib treated groups increased from 2 cell/µL blood on Day 0 to 11 and 51 cells/µL of blood, respectively, by Day 39. The progressive increase of PD-1+ and LAG-3+ percentage cells correlates positively with tumor burden, indicating a dynamic link between biomarker upregulation and the presence of tumor cells in mice. ddPCR analysis revealed an initial CAR copy decrease in all groups, followed by gradual increase in tumor-bearing mice treated with CD19 CAR-T cells, with a dramatic increase observed by Day 39. Results from CAR-T treated and CAR-T plus ibrutinib treated groups were comparable, showing mean copy numbers per ng of DNA increasing from less than 1 on day 4 to 17.9 and 23.3, respectively, on day 39. Importantly, no changes were noted in non-tumor implanted mice, emphasizing the tumor dependency for CAR-T cell expansion. Conclusion: This study emphasizes the marked anti-cancer activity of CD19 CAR-T cells in the NALM6-Luc-mCh-Puro B-ALL murine model. Comprehensive analysis of CAR-T cell persistence and activation, including insights from ddPCR, provides a robust understanding of therapeutic response dynamics. The observed in vivo anti-cancer efficacy aligns with the persistence of CAR-T cells revealed by flow cytometry and the dynamic expansion captured through ddPCR analysis. Our results indicate ddPCR and flow cytometry as complementary and independent methods for assessing the systemic CAR-T cell persistence, contributing to optimizing therapy for B-cell acute lymphoblastic leukemia. Citation Format: Prashant Pandey, David Draper, Sheri Barnes, Yewei Xing, Derrik Germain, Lauren Kucharczyk, Roys Stacey. Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4002.

Targeting autophagy overcomes cancer-intrinsic resistance to CAR-T immunotherapy in B-cell malignancies.

Chimeric antigen receptor T (CAR-T) therapy has substantially revolutionized the clinical outcomes of patients with hematologic malignancies, but the cancer-intrinsic mechanisms underlying resistance to CAR-T cells remain yet to be fully understood. This study aims to explore the molecular determinants of cancer cell sensitivity to CAR-T cell-mediated killing and to provide a better understanding of the underlying mechanisms and potential modulation to improve clinical efficacy. The human whole-genome CRISPR/Cas9-based knockout screening was conducted to identify key genes that enable cancer cells to evade CD19 CAR-T-cell-mediated killing. The in vitro cytotoxicity assays and evaluation of tumor tissue and bone marrow specimens were further conducted to confirm the role of the key genes in cancer cell susceptibility to CAR-T cells. In addition, the specific mechanisms influencing CAR-T cell-mediated cancer clearance were elucidated in mouse and cellular models. The CRISPR/Cas9-based knockout screening showed that the enrichment of autophagy-related genes (ATG3, BECN1, and RB1CC1) provided protection of cancer cells from CD19 CAR-T cell-mediated cytotoxicity. These findings were further validated by in vitro cytotoxicity assays in cells with genetic and pharmacological inhibition of autophagy. Notably, higher expression of the three autophagy-related proteins in tumor samples was correlated with poorer responsiveness and worse survival in patients with relapsed/refractory B-cell lymphoma after CD19 CAR-T therapy. Bulk RNA sequencing analysis of bone marrow samples from B-cell leukemia patients also suggested the clinical relevance of autophagy to the therapeutic response and relapse after CD19 CAR-T cell therapy. Pharmacological inhibition of autophagy and knockout of RB1CC1 could dramatically sensitize tumor cells to CD19 CAR-T cell-mediated killing in mouse models of both B-cell leukemia and lymphoma. Moreover, our study revealed that cancer-intrinsic autophagy mediates evasion of CAR-T cells via the TNF-α-TNFR1 axis-mediated apoptosis and STAT1/IRF1-induced chemokine signaling activation. These findings confirm that autophagy signaling in B-cell malignancies is essential for the effective cytotoxic function of CAR-T cells and thereby pave the way for the development of autophagy-targeting strategies to improve the clinical efficacy of CAR-T cell immunotherapy.

Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

CD19 CAR T-Cell Therapy in Autoimmune Disease — A Case Series with Follow-up

BackgroundTreatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission.MethodsWe evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology–European League against Rheumatism (ACR–EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded.ResultsThe median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR–EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell–associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization.ConclusionsIn this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.)

Clinical features of neurotoxicity after CD19 CAR T-cell therapy in mantle cell lymphoma

AbstractCD19 chimeric antigen receptor (CAR) T-cell therapy has proven highly effective for treating relapsed/refractory mantle cell lymphoma (MCL). However, immune effector cell–associated neurotoxicity syndrome (ICANS) remains a significant concern. This study aimed to evaluate the clinical, radiological, and laboratory correlatives associated with ICANS development after CD19 CAR T-cell therapy in patients with MCL. All patients (N = 26) who received standard-of-care brexucabtagene autoleucel until July 2022 at our institution were evaluated. Laboratory and radiographic correlatives including brain magnetic resonance imaging (MRI) and electroencephalogram (EEG) were evaluated to determine the clinical impact of ICANS. Seventeen (65%) patients experienced ICANS after treatment, with a median onset on day 6. Ten (38%) patients experienced severe (grade ≥3) ICANS. All patients with ICANS had antecedent cytokine release syndrome (CRS), but no correlation was observed between ICANS severity and CRS grade. Overall, 92% of EEGs revealed interictal changes; no patients experienced frank seizures because of ICANS. In total, 86% of patients with severe ICANS with postinfusion brain MRIs demonstrated acute neuroimaging findings not seen on pretreatment MRI. Severe ICANS was also associated with higher rates of cytopenia, coagulopathy, increased cumulative steroid exposure, and prolonged hospitalization. However, severe ICANS did not affect treatment outcomes of patients with MCL. Severe ICANS is frequently associated with a range of postinfusion brain MRI changes and abnormal EEG findings. Longer hospitalization was observed in patients with severe ICANS, especially those with abnormal acute MRI or EEG findings, but there was no discernible impact on overall treatment response and survival.

Adoptive cellular therapy after hematopoietic stem cell transplantation.

Effective cellular therapy using CD19 chimeric antigen receptor T-cells for the treatment of advanced B-cell malignancies raises the question of whether the administration of adoptive cellular therapy (ACT) posttransplant could reduce relapse and improve survival. Moreover, several early phase clinical studies have shown the potential beneficial effects of administration of tumor-associated antigen-specific T-cells and natural killer cells posttransplant for high-risk patients, aiming to decrease relapse and possibly improve survival. In this article, we present an in-depth review of ACT after transplantation, which has the potential to significantly improve the efficacy of this procedure and revolutionize this field.