T-cell Infusion Research Articles

Improved T- and B-Cell Neogenesis in Children with Acute Leukemia Given an Alpha-Beta T-Cell Depleted Haplo-HSCT Combined with the Infusion of Donor T-Cells Genetically Modified with Inducible Caspase 9 Suicide Gene (Rivo-cel)

Background αβ T- and B-cell depleted HLA-haploidentical HSCT (αβhaplo-HSCT) is a suitable transplant option promptly available and virtually applicable to any children in need of an allograft. However, the suboptimal recovery of adaptive immunity still represents a limitation of the approach. In order to better deconvolute the reconstitution of adaptive immunity, in this study we measured T-cell receptor and kappa chain-deleting recombination excision circles (TREC and KREC), which represent reliable surrogates of T and B-cell neogenesis, respectively. We also analyzed a cohort of patients who received after the allograft infusion of a titrated number of donor T-cells genetically modified with inducible caspase 9 (iC9) suicide gene (Rivo-cel) as part of a prospective clinical trial (NCT02065869) with the goal of accelerating the recovery of thymus-independent adaptive immunity. Patients and study design We retrospectively analyzed 134 patients (≤ 25 years) with acute lymphoblastic (ALL) or myeloid leukemia (AML) who received a αβ haplo-HSCT between 2014 and 2021; 65 of them received post-transplant infusion of Rivo-cel, at the dose of 1x10 6 cells/kg, after a median time of 26 days (range: 11-87) post-transplant. No patient received post-transplantation pharmacological GVHD prophylaxis (Locatelli et al. Blood 2017). Details on patient and donor characteristics, as well as on graft composition are reported in Table 1. T-cell neogenesis was investigated by quantifying signal joint- (sj-) and beta (b-) TREC while B-cell neogenesis was analyzed by measuring coding-joint (cj-) and sj- KREC. We performed real-time quantitative PCR, as previously described (Arruda et al. 2018), on genomic DNA extracted from PBMC collected at 6 different time points (before and 1, 3, 6, 12 and 18 months after the allograft). At the same time-points peripheral blood of patients was also evaluated by flow-cytometry to phenotypically characterize the pool of leukocytes. Results With a median follow-up of 6 years (range 1-9) the overall (OS) and disease-free survival (DFS) probabilities of the entire cohort were 88,1% and 82%, respectively. The cumulative incidence (CI) of relapse was 15%. CI of grade II-IV acute Graft-versus-Host Didease (GvHD) was 19%, while that of chronic GvHD (cGvHD) was 7%. In the Rivo-cel cohort, sjTREC and bTREC were consistently detectable as early as one month post-HSCT, while started to recover by 3 months after αβhaplo-HSCT alone, and were significantly higher at +3 ( p=0,0006 and p=0,02, respectively) and +6 ( p=0,0003 and p= 0,001) months post-HSCT as compared with αβhaplo-HSCT. sjTREC and bTREC reached pre-transplant values at 6 and 12 months in Rivo-cel and αβhaplo-HSCT cohort, respectively. B-cell neogenesis, assessed by sj and cjKRECs, showed a superimposable recovery kinetics. A higher number of sjKRECs was observed in Rivo-cel cohort at + 1, +3 and +6 months after transplantation as compared with αβhaplo-HSCT ( p=0,007 , p=0,02 and p=0,04for sjKRECs ; p=0,005 , p=0,092 p=0,035forcjKRECs, respectively) . Furthermore, in the Rivo-cel cohort, patients with detectable sj and bTREC values at +3 months after transplant showed a significantly higher overall survival ( p=0,01 p=0,02, respectively). Also in this cohort, aGvHD was confirmed to play a detrimental effect on T-cell neogenesis, at 3 and 6 months after transplant. The same negative effect was documented for chronic GvHD at +6, +12 and +18 months, also affecting B-cell neogenesis at +6 months after transplant. Type of leukemia did not affect T and B-cell neogenesis. Conclusions In patients given αβhaplo-HSCT, T-cell and B-cell neogenesis were shown to be significantly accelerated by the post-transplant infusion of a titrated number of donor T-cells genetically modified with the iC9 suicide gene. Further studies are needed to elucidate the biologic mechanisms responsible for this favorable effect on recovery of adaptive immunity. Our data confirm the detrimental effect of both acute and chronic GvHD in recovery of thymic function and B-cell neogenesis. In this perspective, the possibility to successfully control/abrogate GvHD through the apoptosis of Rivo-cel triggered by the infusion of the dimerizing agent (AP1903) offers also the advantage of, at least partly, preventing the detrimental effect played by this complication on immune recovery.

Cladribine and Cyclophosphamide Lymphodepletion Prior to Brexucabtagene Autoleucel in Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia and Mantle Cell Lymphoma

Introduction : Fludarabine and cyclophosphamide (Flu/Cy) lymphodepletion (LD) prior to chimeric antigen T-cell (CAR T) infusion favorably impacts CAR T expansion and efficacy. A national shortage of Flu prompted our center to explore substitute Flu with Cladribine (Cla) as part of Cla/Cy LD. Here, we report our experience with this regimen prior to brexucabtagene autoleucel (brexu-cel) for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL). Methods : We performed a retrospective, single-center analysis of consecutive patients who received brexu-cel for RR B-ALL and MCL at Moffitt Cancer Center (1/2022 and 4/2023). The Cla/Cy LD regimen utilized Cy at 500 mg/m 2 x 3 days for MCL or 900 mg/m 2 x 1 day for B-ALL (two patients received MCL dosing) and substituted Flu 30 mg/m 2 with Clad 5 mg/m 2 x 3 days. We compared best overall response (BOR), including measurable residual disease (MRD) status by the clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) ± PCR for B-ALL, event-free survival (EFS, event = progression or death) and overall survival (OS) in those receiving standard Flu/Cy vs Clad/Cy LD. CRS and ICANS were graded as per ASTCT criteria. For patients with a sustained response ≥6 months and who did not undergo stem cell transplant, we assessed serial absolute neutrophil counts (ANC), CD4, CD8, CD19 counts and IgG levels post-CAR T. The HEMATOTOX score (HS, Rejeski et al, 2021) was used to stratify patients for risk of post-CAR T cytopenias. Results : We analyzed 63 patients who received brexu-cel: 23 with ALL (10 Cla/Cy) and 40 with MCL (7 Cla/Cy). Rates of CRS grade ≥ 3 (12% vs Flu/Cy 4%; p=0.29) and ICANS grade ≥ 3 (24% vs Flu/Cy 40%; p = 0.25) did not differ with LD. Two ALL (1 Flu/Cy, 1 Clad/Cy) and one MCL (Flu/Cy) patients died prior to day 28 reassessment. BOR did not differ by LD and histology (Figure 1). MRD negativity in ALL was also comparable (Cla/Cy 5/7, Flu/Cy 10/11, p=0.52). Among ALL patients, 5 received alloSCT consolidation (1 Cla/Cy). Progression was noted in 3/8 Cla/Cy and 6/11 Flu/Cy patients (p=1.0). Fifteen ALL patients experienced early infections (6/10 Cla/Cy, 9/13 FluCy, p=0.69), including bacterial (5), viral (4), and fungal (3). Three deaths (2 Cla/Cy) occurred before day 100, all due to refractory fungemia post anakinra. Two later deaths were attributed to relapse (both Flu/Cy). Among MCL patients, progression was noted in 16 (1 Cla/Cy). Seven MCL patients experienced early infection (all Flu/Cy), including bacterial (6), fungal (2), and viral (1). Two deaths occurred before day 100, 1 related to CRS and fungemia post anakinra (Flu/Cy), and another from stroke (Cla/Cy). Four (all Flu/Cy) have developed a second cancer (2 MDS/AML, 1 prostate, 1 liver), and 1 each died in remission from late infection (sepsis) and intracranial hemorrhage (both Flu/Cy). Cla/Cy patients had higher median ANCs at 28, 90, and 180 days (Table 1). When only patients with HS ≥ 2 were considered, 28-day ANC remained higher after Cla/Cy LD (1.57 vs Flu/Cy 0.67; p=0.04), numerically higher at 90 days (2.36 vs Flu/Cy 1.82; p=0.38) and again significantly higher at 180 days (3.96 vs Flu/Cy 1.2; p=0.05). Median CD4 was higher for Cla/Cy patients at day 28 (0.232 vs Flu/Cy 0.121; p=0.04), and numerically higher at day 90 (0.244 vs Flu/Cy 0.152; p=0.31) and 180 (0.254 vs Flu/Cy 0.161; p=0.21). Median CD8 was similar at day 28 (0.197 vs 0.155; p=0.46), 90 (0.435 vs Flu/Cy 0.167; p=0.65), and 180 (0.557 vs 0.220; p=0.14). There was no difference in IgG levels at day 28 (502 vs Flu/Cy 466; p=0.87), 90 (385 vs Flu/Cy 393; p=0.76), or 180 (424 vs Flu/Cy 426; p=0.94). Median CD19 was greater for Cla/Cy at day 90 (median 0.095 vs 0.000; p<0.01) and numerically higher at day 180 (0.213 vs Flu/Cy 0.000; p=0.29). Median duration of followup was 245d for ALL (FluCy 361d, ClaCy 194d) and 746d for MCL (FluCy 812d, ClaCy 215d). Median DOR, EFS, and OS did not differ by LD (Figure 1). Conclusions : Cla/Cy LD prior to brexu-cel is feasible in ALL and MCL patients with comparable efficacy. Toxicity rates including CRS, ICANS and early infections were similar to those seen with Flu/Cy, but there was a trend towards earlier recovery of neutrophils, B-, and T-cells with Cla/Cy. The trend was demonstrable even in patients at high risk of prolonged post-CAR T neutropenia as identified by the HS. Longer follow-up and correlative analyses are warranted to further explore differences between Cla/Cy and Flu/Cy.

BCMA CAR-T-Cell Therapy in Combination with Long-Term Pomalidomide Is a Safe and Effective Treatment for Relapsed/Refractory Multiple Myeloma

Background: B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor T-cell (CAR-T-cell) therapy has exhibited remarkable efficacy in refractory or relapsed multiple myeloma (R/R MM), but recurrence and rapid progression of disease are still observed in some cases within a short time after treatment. Long-term pomalidomide therapy, which potentiates T-cell functionality and has direct antimyeloma effects, might enhance the efficacy of BCMA CAR-T-cell therapy. Here, we present the efficacy of this combination regimen for R/R MM. Methods: We performed a single-center prospective clinical study that was registered with the Chinese Clinical Trial Registration Center (ChiCTR2000036350). Patients with relpased or refractory multiple myeloma receiving BCMA CAR-T cell infusion were randomized to receive long-term pomalidomide treatment (4 mg/day) one month after infusion. The primary endpoint is overall survival (OS) in R/R MM with long-term pomalidomide administration compared to those without pomalidomide therapy after CAR T-cell infusion. The secondary endpoints include time to progression (TTP) and therapeutic response. Also, the copy numbers of CAR were assessed after infusion. Results: A total of 14 R/R MM patients who received BCMA CAT T-cell therapy, were included in this study. The median age of the enrolled R/R MM patients was 59.5 years (44-71). Five patients had high-risk cytogenetic abnormalities (IGH rearrangement, 1q21 amplification, and P53 deletion) at the time of initial diagnosis. In addition, high-risk genetic alterations were newly detected in patient 2 during first-line therapy. All patients had previously received 3 to 6 lines of treatment with proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), or anti-CD38 monoclonal antibodies. As shown in Figure 1, all patients had demonstrated progressive disease prior to CAR T-cell infusion. The objective response rate (ORR) of BCMA-CART was 100%. Three months following CAR-T-cell infusion, of the 6 patients receiving pomalidomide, all patients (6/6) achieved VGPR (very good partial response) or CR (complete response）, while only 5 patients (5/8) who did not receive pomalidomide treatment achieved VGPR or better. At a median follow-up of 19.7 months, for the 8 patients who did not receive pomalidomide administration, the median TTP (time to progression) was 4.5 (1-14) months, while the TTP was not reached in patients who received long-term pomalidomide treatment. Moreover, the dynamic abundance of circulating BCMA CAR-T cells was monitored as shown in Figure 2. In patients who received pomalidomide treatment after CAR-T-cell therapy, the CAR copy numbers of circulating CAR-T cells peaked 8 to 10 days after CAR-T-cell infusion, with a peak copy number of up to 10 6. In addition, BCMA CAR-T cells persisted (>1*10 3 copies/µg DNA) for 3 months in vivo. Without pomalidomide administration, BCMA CAR-T cells persisted for up to 2.5 months in the peripheral circulation (results not shown). Conclusion: Our results confirm the efficacy of the long-term oral pomalidomide administration (4 mg/day) after BCMA CAR T-cell infusion in patients with R/R MM. Notably, this combination regieme reduced the recurrence rate and prolonged progression-free survival for R/R MM patients, providing a promising option for treating R/R MM. In addition, oral administration of pomalidomide did not significantly prolong the persistence of BCMA CAR T-cells in vivo. However, data from studies with larger sample sizes are required to draw valid conclusions.

Early Progressions of Undetermined Significance (EPUS) in the Month Following CAR-T Cells for B-Cell NHL Is Mainly Related to Pseudoprogression

INTRODUCTION - Relapsed-refractory B-cell non-Hodgkin's lymphomas (R/R-B-NHL) treatment remains challenging. In this context, CAR T-cell therapy has shown sustained efficacy in 40-60% of patients. Early progressions and pseudo-progression have been described after CAR T-cell therapy, but data are scarce. This study aims to assess incidence, risk factors and outcomes of early progression in patients treated with CAR T-cells for R/R B-NHL. PATIENTS & METHODS - All patients were consecutively treated at the University Hospital of Montpellier with a commercial CAR T-cells for R/R-B-NHL between December 2019 and June 2022 and included in the CARTBANK cohort (ID RCB: 2019-A03032-55, NCT04290000). Cases were retrospectively reviewed for early progression. No antitumor treatment was initiated before the day 28 evaluation. Early Progressions of Undetermined Significance (EPUS) were defined as either: self-reported symptom of lymphoma (possible EPUS), clinically assessed progression (probable), or measured through imaging procedure (highly probable) after CAR T-cell infusion and before day 28 (D28) post evaluation. Pseudo-progression was defined as EPUS with partial or complete response on D28 evaluation. On the contrary, hyper-progression was defined as EPUS with confirmed progression on D28 evaluation. RESULTS - A total of 105 patients were included in the study, with a median age of 65 years (interquartile range: 55;70]) and 70% were male. Diffuse large B-cell lymphoma (DLBCL) was the most common histology (89%), followed by follicular lymphoma (6%) and mantle cell lymphoma (5%). Patients had received a median of 2 previous lines of anti-cancer therapy [2;3]. Axicabtagene-ciloleucel was used in 77% of the cases, tisagenlecleucel in 18%, and brexucabtagene-autoleucel in 5%. Overall 18 patients (17%) had EPUS: 2 possible EPUS (11%), 6 (33%) probable EPUS and 10 (56%) highly probable EPUS (panel A). D28 evaluation revealed that most of EPUS were pseudo-progressions (n=13/18, 72%). Hyper-progression was observed in one patient (6%) and 4 patients (22%) died before first metabolic assessment and remained unclassified (panel B). The cause of death was suspected of progression for three of them and sepsis for one. All EPUS occurred before day 21 (median: 7 days [3;8]). Comparison of patients with and without EPUS showed that no clear risk factors for EPUS were identified. EPUS occurrence had no impact on PFS nor OS. The blood expansion of CAR-T cells and their level of exhaustion were monitored by multi-parametric flow cytometry. Regarding these pharmacokinetic parameters, no significant difference was obtained between patients with and without EPUS. Analysis of data regarding total metabolic volume is ongoing and will be presented. DISCUSSION - In this cohort, EPUS is observed in 17% of patients after CAR T-cells infusion for R/R B-NHL. Most of EPUS are related to pseudo-progression. These results should be confirmed in a dedicated study. Thus, early treatment before first evaluation should be avoided.

Updated Follow-up of a Phase 1 Trial of Bispecific CART19/20 Cells for Relapsed or Refractory Non-Hodgkin Lymphoma

CART19/20 was developed by engineering autologous naïve/memory T (T N/MEM) cells with a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR) with the goal of mitigating antigen escape and lack of CAR T-cell persistence. We report an update of an ongoing first-in-human phase 1 clinical trial of CART19/20 for patients with relapsed/refractory non-Hodgkin lymphoma (R/R NHL) (NCT04007029). Eligible patients were ≥18 years old with R/R diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B-cell lymphoma (PMBCL) after ≥2 prior lines of therapy, or with mantle-cell lymphoma (MCL), follicular lymphoma (FL), or CLL/SLL after ≥3 prior lines of therapy. Autologous leukocytes were obtained by leukapheresis and sorted for CD14-/CD25-/CD62L+ T N/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR. Lymphodepletion chemotherapy with fludarabine 30 mg/m 2/day and cyclophosphamide 500 mg/m 2/day was administered on Days −5 to −3 prior to CAR T-cell infusion. The primary endpoint was assessment of safety. Secondary endpoints were overall response rate (ORR), progression free survival (PFS), overall survival (OS), and CART19/20 transgene persistence. As of data cutoff on July 21, 2023, 11 patients had received CART19/20 cells (6 DLBCL, including 4 transformed FL; 3 primary FL; 1 PMBCL; 1 MCL). Patients were treated at two dose levels, either 50 x 10 6 ± 30% CAR+ cells (8 patients) or 200 x 10 6 ± 30% CAR+ cells (3 patients). The median age was 58 (range: 34 to 70). Patients had received a median of 3 prior lines of therapy (range: 2 - 4). None of the treated patients had received prior CAR-T cell therapy; one patient had received a prior CD19-directed therapy. All patients had stage IV disease and 9 of 11 patients received bridging therapy. The engineered CART19/20 cells were enriched with central-memory T cells (T CM: CD45RA-/CD45RO+/CD62L+) which composed a median of 30.8% (range: 5.3 - 80.1%) of the CAR+ T-cells in the infused products. There were no occurrences of CRS grade ≥ 2 or neurotoxicity of any grade. Six patients had grade 1 cytokine release syndrome (CRS) with a median onset of 8 days from infusion (range: 5 - 11 days). One patient met criteria for dose-limiting toxicity with persistent thrombocytopenia after Day+120. Ten of the 11 patients achieved an objective response (91% ORR), with 8 patients (73%) achieving complete response (CR). One patient with FL, who progressed 18 months after receiving CART19/20, received a second infusion of CART19/20 cells and achieved a second CR for 14.9 months. With a median follow-up of 32.3 months (range: 5.1 - 44.0 months), the median PFS (95% CI 2.6 months - not estimable) and median OS (95% CI 5.7 months - not estimable) have not been reached. Overall, CART19/20 demonstrated a favorable safety profile with no severe CRS or neurotoxicity of any grade. Moreover, CART19/20 showed robust activity in patients with R/R NHL with durable responses seen. By utilizing a bispecific CAR and enriching for T N/MEM cells, this phase 1 trial of CART19/20 demonstrates the possibility of improving outcomes through targeting common mechanisms of CAR-T cell resistance.

CAR-T Therapy for the Treatment of Relapsed/Refractory CD19 Positive Large B Cell Lymphoma: Canadian Real-World Evidence

Background: Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable efficacy in relapsed or refractory large B cell lymphoma in clinical trials. However, real-world studies are further needed to determine the feasibility, efficacy, and safety of commercial CAR-T products in a broader population of patients. Current real-world CAR-T therapy studies have focused on large centres, many of whom already had experience in participating in CAR-T clinical trials. Therefore, it remains unknown if similar efficacy and safety outcomes may exist in other countries, like Canada, that have not been treating patients with CAR-T products since their inception. This demonstrates the increasing need for real-world studies in smaller hospital settings now offering commercially available CAR-T products. Methods : We conducted a retrospective review of all patients treated with commercially available CAR-T products, axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel), at The Ottawa Hospital between January 2020 to July 2022. The main objective was to describe the safety and efficacy of the two commercial CAR-T products in a real-world analysis. Results : Fifty-one patients (30 male - 59%), with a median age of 62 (range, 24-82), received commercial CAR-T products: axi-cel (n=27, 53%) or tisa-cel (n=24, 47%). Patient characteristics at infusion were balanced for both CAR-T products in the axi-cel group (68 years old (range, 51-76) vs 61 years old, (range, 24-76, p=0.003). Diffuse large B cell lymphoma (n=33, 65%) represented the majority of the indication for CAR-T therapy, followed by transformed follicular lymphoma (n=17, 33%). Primary mediastinal large B-cell lymphoma was the indication for 1 patient. Patients presented with either relapsed (n=30, 59%) or refractory (n=21, 41%) disease and received a median of 3 (range, 2-7) previous lines of therapy. Patients were referred from 13 centres across Canada and travelled a median distance of 655 km (range 3-3659) to receive treatment in Ottawa. The median time from last progression to referral and consultation was 15 days (range 0-200) and 20 days (range 1-211), respectively. Out-of-province patients experienced significantly longer times to referral and consultation compared to in-province patients (last progression to referral: 9 days versus 34 days, p<0.0001; last progression to consult: 15 days versus 42 days, p<0.0001). The median time from apheresis to CAR T-cell infusion was 36 days (range 26-81), with it being significantly longer for the tisa-cel cohort (p<0.001). Following apheresis, CAR-T products were successfully manufactured for all 51 patients. Out of 49 patients evaluable for response, the overall and complete response rate was 57% and 47%, respectively. With a median follow-up of 219 days (95% CI: 6-722), the median progression-free and overall survival was 257 days (95% CI: 92-NE) and 422 days (95% CI: 106-NE), respectively (Fig. 1). There was a trend towards longer progression-free survival among patients living in-province (280 days, 95% CI: 142-NE) versus out-of-province (115 days, 95% CI: 91-NE) (Fig. 2). Forty-seven patients experienced cytokine release syndrome (grade ≥ 3 in 3, 6%), and 20 experienced neurotoxicity (grade ≥ 3 in 6, 12%). Conclusion : Our results confirm that CAR-T therapy in the Canadian real-world setting has similar outcomes to other published series. Further real-world evidence with more robust patient parameters, like country size and geography, would provide additional crucial insight on distribution and accessibility of CAR-T treatment and demonstrate the importance of national registry data for CAR-T infusions.

Phenotypic, Transcriptomic and Proteomic Characteristics of CAR T-Cell Dysfunction Are Associated with Inferior CAR T-Cell Expansion and Treatment Failure in r/r B-NHL

Introduction: Despite the curative potential of CAR T-cells, a significant number of patients fail to respond or relapse early. Recently, we and others showed that early expansion failure, and particularly CAR T-cell levels at day 7, are strong predictors for treatment resistance (Blumenberg et al, Blood Supplement 2022; Locke et al, Blood Adv 2020). Here we dissected early characteristics of CAR T-cell dysfunction based on immune checkpoint expression, transcriptomic signatures and cytokine profiles and investigated their link to CAR T-cell expansion failure and patient outcome of CD19-targeted CAR T-cell therapy. Methods: Patients with r/r B-NHL who underwent treatment with axi-cel or tisa-cel between January 2019 and November 2021 in the third- or later-line setting at the LMU in Munich were included (n=55). EDTA-anticoagulated peripheral blood and serum was collected. CD19 CAR and immune checkpoint expression (IC; PD-1, TIM-3, LAG-3 and CD224) was assessed by multiparameter flow cytometry on day 14 after CAR T-cell infusion. A next-generation targeted single-cell proteogenomics approach (BD Rhapsody TM) to analyze the transcriptome of distinct CAR T-cell subpopulations on day 7 and day 28 after CAR T-cell infusion. A total of 92 human immuno-oncology related proteins were simultaneously measured using the Olink® Immuno-Oncology panel on day 14 after infusion. Signatures of CAR T-cell dysfunction have been compared between patients with low (loEx) or high (hiEx; < vs ≥ 19 CAR T-cells/µl at day 7 post infusion)CAR T-cell expansion and were put into context of clinical outcome measures. Results: CAR T-cell levels at day 7 were inversely correlated to co-expression of PD-1 (r = -0.4923, p = 0.0011, n = 41), TIM-3 (r = -0.3634, p = 0.0195, n = 41) and LAG-3 (r = -0.5412, p = 0.0003, n = 41) on CAR T-cells at day 14, suggestive of an association of early expansion failure and dysfunction of CAR T-cells. Indeed, loEx (n=26) showed higher frequencies of CAR T-cells coexpressing several IC, including LAG-3 + (p=0.0036), LAG-3 +PD-1 + (p = 0.0048), or LAG-3 +TIM-3 + (p = 0.0065) compared to hiEx (n=15) on day 14 after transfusion. Conversely, we detected higher frequencies of non-dysfunctional CAR T-cell phenotypes negative for several IC, such as CD244 +PD-1 -TIM-3 - (p = 0.0006), CD244 +LAG-3 -PD-1 -TIM-3 - (p = 0.001) and CD244 +LAG-3 +PD-1 -TIM-3 - (p = 0.011) in hiEx. Next, we applied uniform manifold approximation and projection (UMAP) data to examine the surface expression of IC on CAR T-cells at day 14 post-infusion according to treatment response. Unsupervised clustering revealed a higher abundance of CAR T-cell clusters positive for several IC (CD244 +LAG-3 -PD-1 +TIM-3 +) in NR (n = 16) compared to R (n = 23). By contrast, CAR T-cell clusters containing CAR T-cells expressing no IC were more abundant in R compared to NR. In addition, we found significantly increased levels of a highly dysfunctional CAR T-cell phenotype positive for all four IC in NR compared to R at day 14 post infusion (p = 0.0178, n = 46). When specifically comparing the transcriptome of CAR T-cells between NR (n = 6) and R (n = 6), immune inhibitory and exhaustion-related genes were upregulated in NR, such as GZMK, KLRC1 and NR4A2 (figure 1, cluster 1). Conversely, CAR T-cells in R overexpressed genes related to T-cell effector function and cell differentiation, such as TIMP-1, TNFDF10 and MYC (figure 1, cluster 0). Consistent with these findings, a multiplexed proteomics assay for immunomodulatory serum factors in day 14 samples revealed an upregulation of proteins linked to an immune inhibitory and inflamed milieu in patients with highly dysfunctional CAR T-cells (e.g., sPD-L1 and sLAG-3, p = 0.0235 and p = 0.0264, respectively, n = 46) as well as in non-responding patients (e.g., sPD-L1 and IL-6, p = 0.0036 and p = 0.0053, respectively, n = 55). Conclusion: We were able to characterize a phenotypic, transcriptomic and proteomic signature of CAR T-cell dysfunction in post-infusion samples of both patients experiencing CAR T-cell expansion failure as well as lack of radiographic response. Our data show that treatment failure might be predicted not only by CAR T-cell kinetics but also by early assessment of the CAR T-cell phenotype, transcriptome and secretome, and thus enables identification of patients in need of salvage treatment.

Risk Factors and Outcomes for Hematotoxicity Following Tisagenlecleucel for Pediatric B-Acute Lymphoblastic Leukemia

Background: Hematotoxicity following chimeric antigen receptor (CAR) T-cell therapy is increasingly recognized as a distinct adverse event. The CAR-HEMATOTOX score has been developed as a prognostic tool for subsequent development of prolonged cytopenias in adults with lymphoma. However, risk factors in children and young adults (CAYA) with B-acute lymphoblastic leukemia (B-ALL) receiving CAR T cell therapy are less well-defined. We sought to describe incidence, outcomes, and risk factors for prolonged severe neutropenia in CAYA treated with tisagenlecleucel. Methods: This was a multi-institutional retrospective study involving CAYA ≤26 years of age with B-ALL treated with tisagenlecleucel who underwent leukapheresis and manufacturing between August 2017 and March 2020, with data collected through the Pediatric Real World CAR Consortium (PRWCC). Of 185 patients infused, 41 patients were excluded from analysis due to missing data (n=10), refractory disease/death from leukemia (n=27) or non-relapse mortality (n=4) prior to day 35 post-CAR T. Severe prolonged neutropenia was defined as absolute neutrophil count (ANC) <500 cells/mm 3 for ≥30 days. For those with severe neutropenia pre-CAR T, day 0 of severe neutropenia was considered the day of CAR T-cell infusion. Statistical analyses included comparisons using Mann-Whitney and Fisher's exact tests, receiver operating characteristic (ROC) curves to determine cut points with optimal sensitivity and specificity, univariate logistic regression, Kaplan-Meier curves for recovery from neutropenia, overall survival (OS) and relapse free survival (RFS). Results: Of 144 patients, 23 (15.9%) met the definition of severe prolonged neutropenia and 121 (84%) did not. For lymphodepleting chemotherapy, 139 (96.5%) received standard fludarabine (30mg/m 2/dose x 4) and cyclophosphamide (500mg/m 2 x 2). Pre- and post-infusion characteristics are shown in Table 1. Pre-infusion, higher pre-infusion C-reactive protein (CRP), ferritin, bone marrow disease burden, and lower ANC and platelet counts were associated with severe prolonged neutropenia. Post-infusion, any grade cytokine release syndrome (CRS), severe CRS, CRS with hemophagocytic lymphohistiocytosis (HLH)-like manifestations, any grade neurotoxicity, peak CRP and ferritin, use of tocilizumab and steroids, and presence of infections within 30 days were associated with severe prolonged neutropenia. With univariate logistic regression, pre-infusion inflammatory markers, cytopenias, and bone marrow disease burden associated with severe prolonged neutropenia and of these, disease burden >15% had the highest odds ratio (Figure 1). Post-infusion infections and inflammatory toxicities, particularly CRS with HLH-like toxicities, as well as peak CRP, and peak ferritin, were associated with severe prolonged neutropenia. Peak ferritin >3800 ng/ml had the highest odds ratio. In patients with severe prolonged neutropenia, OS was inferior (p<0.0001) and RFS trended towards being inferior (p=0.052), compared to those without. Groups with or without severe prolonged neutropenia were also subdivided into >15% or ≤15% pre-infusion bone marrow disease burden subgroups. OS was similar between patients without severe prolonged neutropenia that had >15% or ≤15% pre-infusion disease burden, but patients with severe prolonged neutropenia and >15% disease burden had inferior OS, suggesting prolonged severe neutropenia may have independent prognostic value. Conversely, RFS was inferior in patients in the subgroups with >15% disease burden compared to those with ≤15%, regardless of occurrence of severe prolonged neutropenia. Discussion/Conclusions: We describe incidence, risk factors and outcomes for the development of severe prolonged neutropenia in CAYA with B-ALL following tisagenlecleucel. While there are similarities in risk factors with the CAR-HEMATOTOX score that has been validated in adults, in CAYA with B-ALL high disease burden pre-infusion and severe inflammation post-infusion were identified as the strongest risk factors for prolonged neutropenia. Identification of risk factors unique to the CAYA B-ALL population allows for tailored risk mitigation in higher risk patients and may help improve outcomes in CAYA at increased risk of prolonged severe neutropenia following tisagenlecleucel.

CD19-Targeting CAR T-Cell Therapy in Transformed Waldenström Macroglobulinemia/Lymphoplasmacytic Lymphoma: A Descar-T and US Collaborative Study

Introduction: Patients with histological transformation (HT) of Waldenström macroglobulinemia (WM) who relapse or are refractory (R/R) have a poor prognosis with standard chemoimmunotherapy, notably patients who are unable to undergo high dose chemotherapy (HCT) and autologous stem cell transplantation (ASCT) or who relapse after HCT/ASCT. CD19-targeted chimeric antigen receptor (CAR) T-cell therapies can lead to durable responses in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and thus may represent a promising therapeutic option in R/R transformed WM (tWM); however, data from pivotal trials is lacking. This study aimed to evaluate the efficacy and safety of anti-CD19 CAR T-cells in real-word patients with R/R tWM. Methods: We retrospectively identified patients with biopsy-proven transformed WM/lymphoplasmacytic lymphoma treated with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) in the French DESCAR-T registry (NCT04328298) and 2 US centers. The primary endpoint was best complete response rate (CRR). Secondary outcomes were best overall response rate (ORR), progression-free survival (PFS), overall survival (OS) and safety. Responses were assessed according to the Lugano 2014 criteria at 1 month, 3 months and 6 months. Subsequent assessments were per institutional pratice. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to the ASTCT 2019 consensus criteria. Hematological toxicity and infections were graded according to the NCI CTCAE (version 5.0). Results: Between June 2017 and May 2023, 22 patients (18 from DECAR-T centers and 4 from US centers) with R/R tWM were treated with anti-CD19 CAR T-cells (13 axi-cel and 9 tisa-cel). Median age at WM diagnosis was 58 years (range 35-72). MYD88 L265P and CXCR4 mutations were present in 85% (11/13) and 29% (2/7) of patients with available data, respectively. Patients received a median of 1 prior line of treatment (range, 0-4) for WM and 2 prior lines (range, 1-4) for HT. Eight patients had received prior ASCT (2 for WM and 6 for HT) and 1 patient allogeneic SCT. The median time from WM to HT diagnosis was 4.5 years (range, 0-32) and 26 months (range, 3-90) from HT diagnosis to CAR T infusion. Of note, CNS involvement was present in 3 patients at DLBCL relapse before CAR T-cell therapy. At time of infusion, the median age was 66 years (range, 41-82) and 8 patients (36%) were older than 70 years. Median Eastern Cooperative Oncology Group (ECOG) was 1 (range, 0-2). Bridging therapy was administered to 16 patients (73%). Following bridging therapy, 6 patients had progressive disease (PD), 4 stable disease (SD) and 6 were responders (2 complete responses (CR) and 4 partial responses (PR)). After CAR T-cell infusion, best CRR was 82% and best ORR was 95%. The ORR/CRR was 95%/77% at 1 month, 76%/71% at 3 months and 68%/68% at 6 months. Seventeen patients (78%) experienced CRS, including 2 grade 3 (all after axi-cel), and 9 ICANS occurred (41%), including 1 grade 3 and 1 grade 4 (all after axi-cel). Eight patients (36%) presented infections, including 3 grade 3 and 1 grade 4. Neutropenia was reported in 78% (grade 3-4 = 68%), thrombocytopenia in 86% (grade 3-4 = 45%) and anemia in 86% (grade 3-4 = 32%) of patients. After a median follow-up of 17 months (range, 1-47), the 1-year PFS and OS were 70% and 84% respectively (figure). Five deaths were reported, 4 due to progressive disease and 1 from SARS-CoV-2 infection. Conclusion: This study shows a high efficacy of anti-CD19 CAR T-cell therapy in R/R tWM with no unexpected toxicity. Longer follow-up is needed to confirm the long-term efficacy of CAR T-cells in tWM.

Evaluation of the T-Cell Metabolic State of Starting Material and Manufactured CAR T Cell Products and Clinical Outcome of CAR T-Cell Therapy for Large B Cell Lymphoma

Background Although chimeric antigen receptor (CAR)-T cells are a viable treatment option for patients with relapsed or refractory (r/r) aggressive B-cell lymphomas, more than half of patients experience relapse within the first two years after product infusion. Successful autologous cell-based anti-cancer therapies require persistence of effector cells, features that highly depend on complex metabolic processes. We earlier demonstrated in chronic lymphocytic leukemia, that expansion and persistence of CD19-CAR T-cell infusion in vivo was correlated with increased mitochondrial mass and enhanced biogenesis(van Bruggen et al, Blood 2019). Metabolic features might therefore serve as early biomarker tools to predict efficacy and outcome of CAR T-cell therapy and might allow therapeutic targeting but did was never studied in a prospective clinical trial. Aims Perform extensive phenotyping and state-of-the-art metabolic state analyses of T cells prior and post CAR T cell production and investigate correlations with clinical outcomes and patient characteristics. Methods Samples were obtained from large B cell lymphoma (LBCL) patients that were part of the TRANSCEND-NHL-001 trial (NCT02631044, Abramson et al, Lancet 2020).Thirty-two patients with r/r B-cell lymphoma who received CAR-T cell treatment lisocabtagene maraleucel (16 responders (R) and 16 non-responders (NR)) were included in our analyses and (metabolically) analyzed by flow cytometry and extracellular flux analysis. Metabolic phenotyping was performed on 53 samples at baseline and 44 samples after an in vitro stimulation. Extracellular flux analysis was done on 14 DP samples. The cohort was split evenly between Responders and Non-responders, and balanced for a score calculated taking key clinical baseline characteristics that were found to be associated with response into account (NCT02631044, Abramson et al, Lancet 2020). Both T cells obtained at time of leukopheresis (starting material (SMAT)) and CAR-T drug products (DP), (following transduction of a CD19-targeted CAR T cell product with a defined CD4 and CD8 composition bearing a 4-1BB costimulatory domain and expansion) were analyzed directly after thawing and after stimulation with anti-CAR-antibody in case of CDP, or αCD3/αCD28 antibodies for SMAT samples. Patients consented to these studies and researchers were blinded for clinical outcomes at time of analysis. Results We found that the baseline clinical factors that associate with clinical response are related to specific metabolic cellular phenotypes, both in SMAT and DP, and with and without T cell stimulation. SMAT samples from patients with higher baseline scores show signs of an increased exhausted phenotype with regards to cellular ROS and exhaustion marker expression, whilst a more plentiful memory-like phenotype was seen in patients with a lower baseline score. When clustering by clinical response status, analysis of glucose and fatty-acid uptake, neutral lipid content, cellular ROS, mitochondrial mass and potential and flux data showed that responders, in both SMAT and DP samples, have an all-round increased metabolic activity upon αCD3/αCD28 or CAR T-receptor triggering, respectively, when compared to non-responders (Figure 1A). Furthermore, responding patients show enrichment of CD27 expression in the SMAT material, indicating a more abundant memory phenotype. Summary/Conclusions This is the first evidence of an association between a specific immune-metabolic phenotype of (CAR-) T cells and outcome of CAR-T cell therapy in patients with relapsed/refractory LBCL. More specifically, a higher abundance of memory phenotypes, metabolic activity and mitochondrial biogenesis of both harvested T cells and CAR-T product associates with improved clinical response. Combining metabolic, phenotypical and clinical characteristics and biomarkers of patient T cells (both pre- or post-production) shows the importance of the metabolic phenotype and fitness of T cell starting material, underlying the importance of early line CAR T therapy following diagnosis.

Lisocabtagene Maraleucel in Relapsed/Refractory Large B-Cell Lymphoma: Real World Analysis from the Cell Therapy Consortium

Introduction Lisocabtagene maraleucel (liso-cel) is an autologous CD19-directed CAR T cell therapy approved for the treatment of relapsed/refractory (R/R) large B-cell lymphoma (LBCL) based on the TRANSCEND (Abramson et al, Lancet 2020), TRANSFORM (Kamdar et al, Lancet 2022), and PILOT studies (Sehgal et al, Lancet Oncol 2022). Extending clinical trial results for liso-cel to the real-world setting, we performed a multicenter retrospective study to evaluate the safety and efficacy of liso-cel in standard of care practice. Methods Patients (pts) aged ≥18 years with LBCL who received commercial liso-cel infusion between February 2021 (time of FDA approval) and June 2023 at 7 academic US medical centers were identified from the Cell Therapy Consortium registry. Pt and treatment characteristics were summarized descriptively, and the Kaplan Meier method was used for survival outcomes. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded per ASTCT consensus criteria. Tumor response was assessed per Lugano criteria locally by the treating clinician. Results As of 6/10/2023, 101 pts underwent liso-cel infusion. Detailed baseline pt characteristics are shown in Table 1. Median age at apheresis was 71 years (range: 30-85) with 35% of pts ≥75 years, 60% were male, 16% had an ECOG PS of ≥2 at apheresis, and 11% had active secondary CNS involvement. By histology, 86% of pts had DLBCL, 7% HGBL, 5% TFL, and 2% PMBCL. Baseline comorbidities included diabetes (18%), stage IV chronic kidney disease (5%), cerebrovascular disease (5%), impaired cardiac ejection fraction (2%), pulmonary dysfunction (2%), impaired hepatic function (2%), and active infection (3%), with 68% having a Charlson Comorbidity Index score of ≥3. Due to comorbidities, 30% of pts would have been ineligible for the TRANSCEND clinical trial. The median number of prior therapies was 3 (range: 1-8) and 16% of pts underwent prior autologous stem cell transplant. Bridging therapy was used in 62% of pts including 41% receiving polatuzumab-based treatment, 24% chemoimmunotherapy, and 21% radiation therapy. Seven (7%) pts received liso-cel on an expanded access trial (NCT04400591) due to a non-conforming product. Median time from apheresis to CAR T-cell infusion was 39 days (IQR: 34-43) and 85% of CAR T-cell infusions occurred inpatient. Any Grade CRS occurred in 49% (3% were Grade ≥3) and any Grade ICANS occurred in 26% (10% were Grade ≥3). Median onset of CRS and ICANS was 4 and 6 days following liso-cel infusion, respectively. Tocilizumab was administered in 31% and 31% of pts received steroids for toxicity management. Nine deaths (9%) unrelated to lymphoma progression occurred at a median of 1.2 months (range: 0.3-4.3) with 2 due to infectious complications, 2 due to other malignancy, 1 due to grade 5 neurologic toxicity, 1 due to concurrent grade 5 neurologic toxicity and grade 5 CRS, 1 due to accidental death, and 2 due to unknown causes. The 6-month incidence of non-relapse mortality was 8% (95% CI, 3.4% - 15%). The overall response rate (ORR) to bridging therapy, as assessed prior to lymphodepletion, was 45% with 17% achieving a complete response (CR). Following liso-cel infusion, response assessment was performed at day 30 and/or day 90 according to treating center practice, or in those determined to have clinical progression. Of the 89 pts evaluable at day 30, the ORR was 81% with 63% achieving a CR. Among 76 pts evaluable at day 90, the ORR was 63% with 58% achieving a CR. With a median follow-up of 10.3 months (range 0.3-22.8), month 6 outcomes were 62.7% (95% CI, 52.8% - 74.3%) for progression-free survival, 71.6% (95% CI, 60.9% - 84.1%) for duration of response, and 77.1% (95% CI, 68.4% - 86.9%) for overall survival ( Figure 1). Conclusions These analyses confirm that efficacy and safety are similar or superior to those of patients enrolled in liso-cel prospective clinical trials for R/R LBCL. Notably, these outcomes were achieved in pts predominately of advanced age and with a significant comorbid burden, recognizing that one-third would have been ineligible for TRANSCEND. These results also likely reflect advancements in patient selection and toxicity management including the real world utilization of novel bridging therapy to temporize and debulk disease. Follow-up is ongoing and updated data will be presented at the meeting.

Early Autologous Hematopoietic Stem Cells (AHSC) Boost (HCB) to Overcome Immune Effector Cell-Associated Hematotoxicity (ICAHT) after CAR T-Cell Therapy for Lymphoma: A Single Center Experience

Sustained cytopenias designed as Immune Effector Cell-Associated Hematotoxicity (ICAHT) represent one of the most frequent side effect of CAR-T-cell therapy (30 to 90 % patients in recent real life studies) resulting in significant morbidity, mortality and resources consumption. We report single center experience of early reinjection of previously cryopreserved AHSC (HCB) in that setting. From July 2021 until January 2023, 27 patients received CAR T-cell therapy (25 Axi-Cel, 2 Brexu-Cel) after Fludarabine and Cyclophosphamide lymphodepletion for relapsed/refractory large B-cell lymphomas (24), mantle lymphoma (2), or follicular lymphoma (1). Median age was 62 years (19-75). CAR-HEMATOTOX score was ≥ 2 for 46% of patients. All patients received Filgrastim from D0. Hematopoietic recovery was longitudinally evaluated until D90 or censoring for relapse and/or subsequent cytotoxic treatment: severe (CTAE grade ≥ 3) neutropenia, anemia or thrombopenia occurred respectively for 88, 44 and 76% of patients until D28, and 60, 24 and 60% until D90. Median duration of severe neutropenia was 7 days (0-39) and 28 days for severe thrombocytopenia (0-118). Prolonged neutropenia occurred in 18/25 evaluable patients, mostly with a biphasic course (intermittent recovery and aplastic phenotype for 14 and 4 patients respectively). CAR-HEMATOTOX score was significantly correlated with sustained thrombopenia (p = 0,005) but only marginally with ICAHT (p = 0,054) and not with severe neutropenia (p = 0,44) . We observed significant correlation of severe ICAHT with progressive disease at CAR T-cell infusion (p = 0,015), platelets nadir < 30 giga/l before D28 (p < 0,005) and CAR T-cell expansion (> 200/µl) at D7 (p = 0,023). 7 patients received HCB (no conditioning) at a median D24 (15-41) post CAR T-cell infusion: after D14 for patients with aplastic phenotype, or for cytopenic recurrence for patients with biphasic course. Filgrastim was pursued or reintroduced until neutrophil recovery (> 1 giga/l) which occurred after a median of 10 days (4-16); platelet recovery (> 20 giga/l and > 50 giga/l) occurred after a median of respectively 12 (8-14) and 17 (11-22) days. If we consider D0 as CAR T-cell infusion, neutrophil recovery occurred at median D37 (22-49) and platelet recovery (> 50 giga/l) at median D37 (26-63). Median complete hematopoietic reconstitution (defined as PMNC > 1 giga/l, and Hb > 100 g/l, and PLT > 100 giga/l) was significantly shorter (median 23 days, 14-30) for patients receiving HCB compared with patients with prolonged cytopenia (> 28 days) who do not receive HCB (60 days, p = 0,018). Transfusion and hematopoietic growth factors (HGF) consumption was significantly reduced after HCB but we observe no difference between 2 groups considering hospitalization, EFS or OS. 2 patients receiving HCB experienced late recurrence of cytopenia (after D90) requiring intermittent HGF support but no transfusion or hospitalization. Discussion - Perspectives Recent case series highlighted clinical feasibility and safety and of this approach across a broad population of pediatric and adult patients: we validate this strategy in a more homogeneous and systematic practice. As study population is small, we failed to demonstrate that earlier application of an HCB is associated with superior survival outcomes as shown by others. We agree with recent updated recommendation of experts panel considering application of HCB without prior conditioning chemotherapy for grade ≥ 3 ICAHT beyond D14 (so considered early) if AHSC graft is readily available. Prophylactic AHSC harvest in high-risk candidates has been proposed but collection process may add to already high logistic burden and costs of CAR T-cell therapy in the lymphoma setting. Conversely, cryopreserved ASHC is mainly available for patients with myeloma and this approach would be readily proposed and useful for around 20% patients as recently described. Gagelmann N et al. Hematopoietic stem cell boost for persistent neutropenia after CAR T-cell therapy: a GLA/DRST study. Blood Adv. 2023;7(4):555 Rejeski K et al, Immune Effector Cell-Associated Hematotoxicity (ICAHT): EHA/EBMT Consensus Grading and Best Practice Recommendations. Blood. 2023 doi: 10.1182/blood.2023020578 Davis JA et al. Efficacy and safety of CD34+ Stem Cell Boost for Delayed hematopoietic Recovery After BCMA Directed CAR T-cell Therapy. Transplant Cell Ther. 2023 doi: 10.1016/j.jtct.2023.05.012

Early Prediction of Treatment Response By Circulating Tumor DNA Profiling in Patients with Diffuse Large B-Cell Lymphoma Receiving CAR T-Cell Therapy

Introduction Patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) are characterized by a particularly poor prognosis. Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has improved outcomes of patients with r/r DLBCL, yet a substantial proportion of patients still experiences relapse or progression after CAR T-cell treatment. Identifying patients at high risk of CAR T-cell therapy resistance or future lymphoma progression remains challenging. Here, we explored the value of circulating tumor DNA (ctDNA) as a prognostic biomarker at baseline and early into treatment in DLBCL patients undergoing standard-of-care CAR T-cell therapy, without the need for matched tumor genotypes. We further investigated associations between ctDNA and other known clinical risk factors. Methods We applied targeted next-generation sequencing (NGS) to a total of 53 samples from r/r DLBCL patients receiving CAR T-cell therapy (axicabtagene ciloleucel and tisagenlecleucel, n=16) at the University Medical Center Freiburg (Germany), using a modified version of the AVENIO ctDNA analysis workflow (Roche; Research Use Only) that covered ~314kb and targeted 466 distinct genes recurrently mutated in DLBCL (based on the CAPP-Seq workflow, Kurtz DM et al. J Clin Oncol 2018). Tumor genotypes were assessed noninvasively from plasma samples obtained at baseline before lymphodepletion with matched germline controls. Concentrations of ctDNA were quantified at baseline and early after CAR T-cell therapy at day 7-10. Initial treatment response was assessed by conventional PET-CT obtained 4-6 weeks after treatment based on the Lugano criteria. Results At baseline prior to lymphodepletion, we detected somatic mutations in 100% of plasma samples by noninvasive genotyping, with a median of 19.5 mutations per patient (range: 3-97). The most frequently mutated genes were IGHV, IGLL5, BCL2, MYC, BTG1, CREBBP, and TP53. Patients with high baseline ctDNA concentrations (mean allele frequency [AF] >= 1%) showed shorter progression-free survivial (PFS) and overall survival (OS) than patients with ctDNA levels below this threshold (median PFS: 86.5 days vs. not reached, p=0.04, HR: 6.3, 95% CI: 1.7-23.6; median OS: 219 days vs. not reached, p=0.048, HR: 4.1, 95% CI 1.0-16.6, Figure 1). Pretreatment ctDNA levels were significantly correlated with LDH concentrations ( p=0.049, r=0.5). We further found significantly higher ctDNA concentrations in patients with transformed DLBCL (vs. non-transformed, p=0.008) and in those with 3 or more prior treatment lines (vs. 1-2, p=0.04). At day 7-10 after CAR T-cell therapy, ctDNA levels dropped a median of 10-fold compared to the pretreatment time point ( p=0.008). Patients achieving a complete (CMR) or partial metabolic response (PMR) by PET-CT after 4-6 weeks showed a significant decrease of ctDNA ( p=0.001), while those with a stable (SD) or progressive disease (PD) had no significant ctDNA reduction at day 7-10 after CAR T-cell infusion. Vice versa, while 100% of patients with a >1.5-log-fold drop of ctDNA level between the pretreatment and interim time point revealed a CMR or PMR in early PET-CT scans, only 62% of patients with a smaller ctDNA decline responded to treatment according to PET-CT. Conclusions Our data suggests, together with previously published studies (Sworder BJ et al., Cancer Cell, 2023; Frank MJ et al. J Clin Oncol, 2021), that ctDNA profiling may allow robust noninvasive genotyping, accurate risk-stratification, and early prediction of clinical outcomes in r/r DLBCL patients undergoing CAR T-cell therapy. Although limited by small sample size, this pilot study highlights a potential future role of ctDNA measurements for personalized treatment selection and guidance in clinical trials.

Health-Related Quality of Life Profile and Symptom Burden of Patients with Aggressive B-Cell Lymphomas Just before CAR-T-Cell Therapy: A Real-World Study By the Gimema

Background Although efficacy and safety data on chimeric antigenic receptor (CAR) T-cell therapy for patients aggressive B-cell lymphomas are being accumulated, there is limited research on health-related quality of life (HRQoL) and symptom burden of these patients. Additionally, the few HRQoL data available mainly stems from selected patients enrolled in clinical trial settings. Objective The primary objective of this study was to compare HRQoL of patients with relapsed/refractory aggressive B-cell lymphomas just before receiving CAR T-cell infusion in real-life, with that of their peers in the general population. Secondary objectives were to describe the prevalence of disease-specific symptoms and worries, and to investigate the relationships between symptom burden and HRQoL and functional outcomes. Methods: Baseline data from an ongoing prospective observational study by the Italian GIMEMA Group were analyzed. Inclusion criteria were: adult patients with diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma, and DLBCL transformed by indolent lymphoma and mantle cell lymphoma, scheduled to receive CAR T-cell therapy. HRQoL was assessed with the EORTC QLQ-C30 and its recently validated module for patients with high-grade non-Hodgkin lymphoma (EORTC QLQ-NHL-HG29). Multivariable linear regression analyses were performed to estimate the overall mean difference in the QLQ-C30 scores between patients and the general population, adjusting for age, sex and comorbidity. Evidence-based guidelines for interpretation of the QLQ-C30 were used to determine clinically meaningful differences between groups. The prevalence of patients who reported symptoms and worries on the QLQ-NHL-HG29 was based on the number of patients who answered, “a little,” “quite a bit,” or “very much” on a given item. This was assessed overall and by age groups. For descriptive purposes, we divided the sample by the median age, deriving two subgroups: younger (with a median age of 50 years; IQR: 41 - 56) and older patients (with a median age of 65 years; IQR: 63 - 71). Mean scores of functional and global QoL scales of the EORTC QLQ-C30 were analyzed by severity of symptom burden. Patients were categorized as having high or low symptom burden based on the median score of the validated symptom burden scale of the QLQ-NHL-HG29 questionnaire. Results Overall, 65 patients enrolled across 9 centers were analyzed. Median age at study entry was 61 years (IQR, 50-65) and 21 (32%) were women. Median time since diagnosis was 2 years (IQR, 1-4). Eighteen patients (28%) had already received autologous hematopoietic stem cell transplantation (auto-HSCT) before study inclusion. Median number of previous lines of therapy was 2 (IQR: 2-3), and eleven patients (18%) reported at least 1 comorbidity. Patients reported statistically and clinically meaningful worse scores across several domains, compared with their peers in the general population. The top three largest statistically and clinically meaningful differences with regard to functional scales were observed for role functioning (Δ=26.8, p<.001), social functioning (Δ=17.6, p<.001) and the global QoL scale (Δ=14.3, p<.001). With regard to symptoms, the two largest statistically and clinically meaningful worse symptoms, compared to the general population, were observed for fatigue (Δ=14.2, P<.001), dyspnea (Δ=10.9, p=.003) and financial difficulty (Δ=10.6, p=.001). Prevalence of symptoms and worries was high with more than 50% of patients reporting 14 different symptoms and worries. Younger patients tended to report a higher prevalence in regard to specific worries. The top ten most prevalent symptoms and worries by the QLQ-NHL-HG29, overall and by age groups, are reported in Table 1. Patients with a greater symptom burden had a clinically meaningful worse global QoL, and worse physical, role, social, and cognitive functioning compared to those with a lower symptom burden (Figure 1). Conclusion: Using a validated disease-specific measure for high-grade non-Hodgkin lymphoma, we observed that just before CAR T-cell therapy in real-life, patients report substantial HRQoL impairments and a high prevalence of symptoms and worries. Our findings also suggest to pay special attention to patients with a greater symptom burden, being a vulnerable group with important HRQoL and functional impairments.

Reduction of Metabolic Active Tumor Volume Prior to CAR T-Cell Therapy Improves Survival Outcomes in Patients with Large B-Cell Lymphoma

Introduction CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has become the standard 2 nd or 3 rd line therapy for patients with relapsed or refractory Large B-cell lymphoma (r/r LBCL). As part of this treatment bridging therapy is frequently used between T-cell apheresis and CAR T-cell infusion in order to limit disease progression, diminish lymphoma-related symptoms and/or reduce tumor volume. Bridging therapy has shown to be feasible, safe and effective in patients prior to infusion. In addition, a lower tumor volume is associated with better treatment outcomes and less toxicity. However, the exact reduction and dynamics in metabolic active tumor volume (MATV) are unknown. Here we investigated the relation of changes in pre-apheresis (baseline) MATV, based on 18F-FDG PET scans, and pre-lymphodepleting chemotherapy (pre-LD) MATV with CAR T-cell outcomes and toxicities in patients with r/r LBCL. Methods Baseline and pre-LD MATVs and PET characteristics were calculated semi-automatically of patients who were treated with CAR T-cell therapy. A Standardized Uptake Value (SUV) threshold of 2.5 was used to maintain robustness and avoid MATV underestimation. Bridging strategies consisted of radiotherapy, chemotherapy, or a combination of both. Primary endpoints were duration of response (DOR) and overall survival (OS). Secondary endpoints included cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The Youden index was used to identify the optimal cut-off for dividing patients into low- and high-MATV groups to predict DOR at 2 years. Two-year DOR and OS rates were estimated using Kaplan-Meier curves and the log-rank test was utilized to compare the differences among low- and high-MATV patient groups. Univariable analysis was performed with Cox regression for DOR and OS, and with logistic regression for CRS and ICANS. Results Seventy-four patients with LBCL and treated with CAR T-cell therapy were included in the study. For all patients baseline 18F-FDG PET scans were available, whereof in 68 patients the pre-LD scans. The median baseline MATV was 180 cc (IQR 64 - 426) with a median SUV max of 18.8 (IQR 11.9 - 28.1) and increased to 241 cc (IQR 75 - 525) at pre-LD, with a median SUV max of 21.0 (IQR 12.8 - 31.3). The optimal cut-offs for low- and high-MATV patient groups at baseline and pre-LD were 190 cc and 480 cc, respectively. Patients with a low MATV baseline had a better DOR (Figure 1A) and OS compared to high MATV baseline patients (both p < 0.001). Patients with a high MATV baseline that was reduced to a low MATV pre-LD showed a significant improvement in DOR (Figure 1B) and OS, compared to patients with a high MATV pre-LD ( p = 0.036 for DOR; p = 0.015 for OS). Univariable analysis showed that a high baseline MATV was associated with a worse DOR (HR 6.43 CI [2.15 - 19.20]; p < 0.001) and OS (HR 6.97 CI [2.33 - 20.80]; p < 0.001). A high pre-LD MATV was also associated with a worse DOR (HR 4.86 CI [1.98 - 12.00]; p < 0.001) and OS (HR 5.87 [2.41 - 14.30]; p < 0.001). Other significant associations with DOR and OS were observed for pre-LD lactate dehydrogenase (LDH), the IPI score for DOR exclusively, and gender and bridging therapy for OS exclusively. Conclusions We showed that patients with a low MATV compared to a high MATV at baseline have better survival outcomes and less toxicity. Additionally, reducing tumor volume by applying bridging therapy in patients with a high baseline MATV, results in a significant better DOR and OS. Reducing the MATV before CAR T-cell infusion is therefore essential in CAR T-cell therapy patient care and provides a rationale for the use of more intensive bridging therapies.

CAR-T Phenotype and Efficacy Is Impaired By Lymphoma-Associated CD39 + T Cells

Background: Autologous chimeric antigen receptor (CAR) T-cell therapy is standard of care for patients with relapsed or refractory Large B cell Lymphoma (R/R LBCL). Each patient's CAR T product is unique, and the patient-specific determinants of CAR T-cell quality are poorly understood. CD39 is an ectonucleosidase that participates in the conversion of ATP and ADP and is upregulated in cancer. On T-cells, CD39 is expressed on CD4+ Tregs and on exhausted CD8+ T-cells that are prone to apoptosis. Methods: This is a retrospective single-center cohort study (N=72) of patients who received CD19 CAR T-cell therapy for LBCL (axi-cel n=52; tisa-cel n=20). Leukapheresis material was obtained from n=51 patients and flow cytometry was performed to phenotype CD4+ and CD8+ stem-central memory, memory, and effector T-cell subsets (using CCR7 and CD45RO), and for immune checkpoint expression (PD-1, LAG3, TIGIT, and CD39). 10x multiome (ATAC and RNA) single cell sequencing (scRNA-seq) was performed on n=8 leukapheresis products for deeper characterization. Serum metabolomics was performed on paired samples from n=21 patients. 10X Genomics Chromium scRNAseq was also performed on n=57 CAR T-cell infusion products, including n=36 with paired leukapheresis material previously phenotyped by flow cytometry. In vitro CAR T-cells were manufactured from starting patient T-cells and analyzed for CAR T-cell phenotype and function. Results: In the leukapheresis material of patients that did not achieve long-term remission after CAR T-cell therapy, and in those with high tumor burden, we found higher numbers of CD4+CD39+ and CD8+CD39+ T-cells ( Figs. 1 and 2). 10X multiome sequencing confirmed that CD39+ T-cells exhibit characteristics of exhaustion. Recent platinum-based chemotherapy did not affect CD39+ T-cell levels. In n=21 patients with paired serum analyzed by metabolomics, high CD8+CD39+ T-cells associated with reduced metabolites in the inosine-hypoxanthine pathway, which are downstream of the ectonucleotidase function of CD39 (inosine; P=0.006; hypoxanthine P=0.07). Increased numbers of CD39+ T-cells in the starting leukapheresis material translated into manufactured CAR T-cells having less favorable product characteristics, including higher CD39 expression and fewer memory cells in both paired patient samples and in experiments of in vitro CAR T-cell manufacturing. ScRNA-seq of the infused CAR T-cell product revealed differential gene signatures, both globally and in association with response, between axi-cel and tisa-cel CAR T-cell products, highlighting the profound product-specific differences. Nonetheless, both products were adversely affected by high CD39+ T-cells in the leukapheresis material used for manufacture. Conclusions: CD39+ T-cells, found in patients with high lymphoma tumor burden and an unfavourable immunometabolic environment, associate with poor CAR T-cell quality and adverse patient outcomes in R/R LBCL. Strategies are needed to improve patient T-cell quality prior to leukapheresis and/or improve CAR T-cell manufacturing from patients with high levels of exhausted CD39+ T-cells. Figure 1: Progression-free survival (PFS) stratified by leukapheresis CD8+CD39+ T-cells in the leukapheresis product. Blue - below cohort median percentage of CD8+T-cells that are CD39+ (low); Red - above median percentage of CD8+T-cells that are CD39+ (high). P-value by Log-Rank test. Overall survival (not shown) P-value 0.01. Figure 2: Proportion of CD8+CD39+ T-cells found in patients with low or high baseline metabolic tumor volume (MTV) based on a previously established cutoff (Dean et al. Blood Adv. 2020). MTV was measured on the pre-CAR T-cell PET/CT. P-value by T-test. MDJ, XY, and RMA are co-first authors with equal contribution.

Patterns of Use and Impact of Post-Referral and Bridging Therapy on Outcomes in Patients Undergoing CAR T-Cell Therapy for B-Cell Lymphoma: A Single-Center Analysis

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has changed the treatment landscape for B-cell non-Hodgkin lymphoma (B-NHL) . Treatment is often given after patients have been referred for CAR T-cell therapy while evaluation is underway but prior to leukapheresis, subsequently referred to as post-referral therapy (PRT). Bridging therapy (BT) may be used while CAR T cells are being manufactured. Little has been previously reported on patterns of use of various regimens, optimal choice of treatment, and how use of these treatments impacts patient outcomes when undergoing CAR T-cell treatment. METHODS: Patients aged ≥18 years with B-NHL who underwent apheresis between November 2017 and March 2023 for commercial CAR T-cell therapy at Vanderbilt University Medical Center were identified from an internal database. Patients received axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), or brexucabtagene autoleucel (brexu-cel). The primary endpoint was complete remission (CR) at 30 days post CAR-T. Patients who died prior to that time point were not censored and considered to not have achieved CR. Tumor response after CAR T-cell infusion was assessed per Lugano criteria by the treating clinician. PRT and BT regimens were defined as follows: platinum-based included cisplatin, carboplatin, or oxaliplatin; polatuzumab-based included rituximab with or without bendamustine; non-platinum based included: R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), R-hyper CVAD (rituximab, hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone and/or high-dose methotrexate and cytarabine), or R-mini CVD (rituximab, cyclophosphamide, vincristine, dexamethasone); oral targeted agents included: ibrutinib, venetoclax, or lenalidomide based treatments. Differences in baseline characteristics were assessed with chi squared analysis and two sample t-test. Predictors of 30-day CR were identified with logistic regression analysis. RESULTS: This study included 60 patients who were treated with commercial CAR-T with median follow up of 280 days. A total of 9 did not receive any PRT while 51 received one or more lines of PRT. There were no statistically significant differences between those who did or did not receive PRT in median age, sex, diagnosis, disease stage, or product type (Table 1). Elevated LDH (44% vs. 80%, p=0.022) and private insurance (33% vs. 69%, p=0.043) were more common in those who received PRT compared to those who did not. There were no significant differences in the receipt of BT. A detailed summary of types of regimens used in PRT and BT is included in Table 1. For those who received PRT, the median total number of cycles of treatment received including bridging therapy was 3 (range 1-7). The median time from referral to leukapheresis was longer in those who received PRT compared to those who did not (68.5 days vs. 36 days). Fewer patients who received PRT underwent leukapheresis within 45 days (22% vs. 56%, p=0.037). There was no difference in 30-day CR after CAR-T infusion. Figure 1 shows 30-day CR stratified by type of PRT given in the first line. Amongst patients who received platinum-based therapy, 12 (60.0%) achieved a CR at 30 days, while 8 (40.0%) did not. Amongst patients who received polatuzumab-based therapy, 4 (26.7%) achieved a CR at 30 days, while 11 (77.3%) did not. In univariate logistic regression analysis, predictors of achieving CR at 30 days included age (OR 1.04, CI: 1.00-1.09, p=0.033), elevated LDH (OR 0.19, CI: 0.051-0.69, p=0.012), and receipt of platinum-based therapy as first line PRT (OR 4.13, CI: 1.23-13.78, p=0.021). Multivariate analysis showed receipt of platinum-based therapy as first line PRT (OR 5.11, CI: 1.23-21.22, p=0.025) and elevated LDH (OR 0.08, CI: 0.013-0.53, p=0.09) were significant predictors. CONCLUSION: In this retrospective analysis, we provide a detailed overview of the number of lines and types of PRT and BT used in patients undergoing CAR-T cell therapy at a single US academic medical center. While 30-day CR was not impacted by the receipt of PRT, patients who received platinum-based chemotherapy as their first PRT had significantly higher odds of achieving of 30-day CR than those who received other therapies in multivariate analysis. This trend will be important to explore in larger analyses and may provide insight into optimal post-referral treatment choices.

Low Tmtv Influences Response and Outcomes in R/R DLBCL 3L+ Patients Treated with CAR-T Cells : First Results of FDG-PET/CT Analysis in the French Descar-T Registry

Context : DESCAR-T is a French national registry designed by LYSA/LYSARC including real-life patients receiving commercial CAR T-cells for hematologic malignancies. We initiated a project to conduct a centralized review of Fluorodeoxyglucose Positron Emission Tomography and Computed Tomography (FDG-PET/CT) performed before CAR T-cells infusion, and during follow-up, to evaluate the impact of several metabolic imaging parameters at baseline and response assessment. Materials and methods : Patients with relapsed/refractory (R/R) DLBCL who initiated at least a third line of therapy (3L+) from the DESCAR-T registry and treated with commercial anti-CD19 CAR T-cells, with at least one central reviewed PET before CAR T-cell infusion (PRE-CAR T), at one month (M1) or three months (M3) after infusion, were included. For each visit, Total Metabolic Tumor Volume (TMTV) and SUVmax of the tumor were measured. Deauville score (DS) and response according to 2014 Lugano classification were registered on follow-up FDG-PET/CT. TMTV optimal cut-off for survival prediction (PFS and OS) was determined using X-tile analysis and association of DS with prognosis was studied. Progression-free survival (PFS) was defined as the time between CAR T-cell infusion and progression during follow-up. Overall survival (OS) was defined as the time between CAR T-cell treatment and death during follow-up. Results : Our analysis included FDG-PET/CT data from 212 R/R DLBCL patients (59 treated with tisagenlecleucel and 153 with axicabtagene ciloleucel). Median follow-up was 24.1 months and overall response rate was 47.1% with 112 patients presenting progression or relapse. On PRECAR-T FDG-PET/CT, the median SUVmax was 16.1 (25th-75th percentiles, 8.55-25.30) and median TMTV was 43.91 cm3 (25th-75th percentiles, 12.79-140.43 cm3). Tumor burden significantly affected survival using a TMTV optimal cut-off of 30 cm3. It was associated with a shorter PFS in patients with PRECAR-T TMTV ≥30 cm3 compared with <30 cm3 (median of 3 vs 22.2 months; p=0.0001; respectively) and a shorter OS (median of 9.4 vs 32 months ; p=0.0001; respectively). Regarding metabolic response at M1, reported in 192 patients, DS 1-3 (n=120; 62.5%) was significantly associated with better PFS and OS than DS4-5 (n=72; 37.5%) (for PFS: median of 1.3 vs 21.8 months ; p<0.0001; for OS: median of 4.5 vs 30.6 months; p<0.0001). Of note, considering only DS5 (n=46; 24%) as unequivocally insufficient response onM1 evaluation, identified patients with the poorest outcomes (for PFS: median of 0 month vs 21.2 months ; p<0.0001; for OS: median of 4.5 months vs 31.1 months; p<0.0001). Conclusion : Low PRECAR-T TMTV influences response and retains its predictive and prognostic value in R/R DLBCL patients treated with anti-CD19 CAR T-cells. Moreover, metabolic response assessed with DS is significantly associated with patients' outcome.

Invasive Fungal Infections after CAR T-Cell Therapy for B-Cell Lymphoma: A Study from the French Descart Registry

Introduction: Chimeric antigen receptor (CAR) T cell therapy has emerged as an important treatment option for patients with B-cell malignancies. Despite its major clinical impact, CAR T cell therapy holds a potential risk of increased susceptibility to infections, including invasive fungal infections (IFI). Since data about the prevalence and impact of fungal infections in this population are limited, we aimed to provide a comprehensive overview of IFI using real-world data from the national DESCAR-T registry. Methods: We performed a multi-center retrospective cohort study, quering the French DESCAR-T registry (NCT04328298), to describe type, frequency and outcomes of IFI in adults with refractory or relapsed (R/R) B cell lymphomas. In addition, healthcare providers were contacted to declare any missing cases. Demographic, biological and clinical characteristics were collected retrospectively. Each IFI case was reviewed by an infectious disease specialist and a microbiologist, and classified according to the European Organization for Research and Treatment of Cancer (EORTC) criteria (Donnelly et al. Clinical Infect Dis 2020). Results: Among the 1144 patients included in the registry from 2018 to 2022, we identified 57 suspected IFI cases. After exclusion of 25 patients not adjudicated for IFI, 32 patients (3%) were identified as having presented a proven (n = 14), probable (n = 12), or possible (n = 6) IFI. DLBCL was the primary diagnosis for 30 patients and axi-cel was the most frequent CAR T product (23/32, 72%). Seven patients (22%) had received a previous hematopoietic stem cell transplantation. All patients experienced cytokine release syndrome (including 8 grade ≥ 3) and 21 (66%) experienced immune effector cell-associated syndrome (including 13 grade ≥ 3). Overall, 27 (84%), 21 (66%), and 5 (16%) received tocilizumab (median 960mg, range 120-2880mg), corticosteroids, and anakinra, respectively. Among known risk factors for IFI, grade 4 neutropenia and late neutropenia (< 1g/L) at day 28 after infusion were identified in 25 (78%) and 14 (44%) patients respectively. Overall, 8 (25%) patients received antifungal prophylaxis, posaconazole was used for 4 of them. Over half of the IFI cases occurred between 0 and 30 days after CAR T-cell infusion (18/32, 56%), with a median onset time of 29.5 days (IQR 16.5 - 79.5). The most frequently observed infections were invasive Aspergillosis (n = 13), with pulmonary involvement in 10/13 (77%) of patients, leading to a fatal issue in 3/13 patients (23%). Other mold infections included Mucormycosis (n = 5), with pulmonary involvement in 4/5 patients and a fatal outcome in 1/5, as well as Fusariosis (n = 1). Yeast infections (n = 12) were mostly identified as Candidemia (92%) with a mortality rate of 3/12 (27%). The median overall survival of patients with IFI was 186.5 days (IQR 79-506), 7 deaths being related to IFI and 3 to lymphoma relapse. Median progression-free survival was 94 days (IQR 31 - 252). Conclusions: This study represents one of the largest investigations about IFI in B-cell lymphoma patients treated with CAR T-cells. Even though IFI represent a rare complication after CAR T-cell therapy, the related-mortality is high. Selective antifungal prophylaxis may benefit high-risk patients, while rapid preemptive diagnostic workup, particularly for lung involvement, is mandatory. Analysis to define potential risk factors for IFI are ongoing.

Second-Line Chimeric Antigen Receptor T Cell Therapy (CAR-T) As Standard of Care for Relapsed-Refractory Large B-Cell Lymphoma (LBCL)

Introduction: The randomized registrational ZUMA-7 (Locke, NEJM 2022) and TRANSFORM (Kamdar, Lancet 2022) trials in second line (2L) LBCL showed superiority of CAR T when compared to standard of care (SOC) chemotherapy-based regimens with a goal of consolidative autologous stem cell transplant. These results led to the FDA approval of axicabtagene ciloleucel (axi-cel) and lisocabtagene maraleucel (liso-cel) in 2L for LBCL with refractory disease or relapse within 1 year of completing induction chemotherapy. Significant differences exist between the registrational trial patient population and the real-world commercial CAR-T recipients in 3L+ setting (Nastoupil, JCO 2020, Jacobson, JCO 2020). Herein, we describe the outcomes of patients with LBCL treated with 2L commercial CAR T therapy. Methods: Five US academic institutions contributed data to this retrospective study. CAR T associated toxicities were graded by ASTCT consensus grading criteria (Lee, TCT 2019) and investigator assessed responses were reported per Lugano 2014 criteria. Patients who received CAR T therapy in 2L after the histologic diagnosis of LBCL were included, with leukapheresis between SOC approval on 4/1/2022, and 4/25/23. Patients were included if they received intermediary therapies before CAR T-cell infusion. These intermediary therapies included disease-holding therapy, defined as therapy given between relapse and the time of leukapheresis, given with the intent to “hold” patients' disease under control until CAR T-cell therapy was arranged (Bucklein, Hemasphere 2023). Patients who received therapy after relapse with the intent of restaging to decide on subsequent CAR T-cell therapy were excluded. Patients who received bridging therapy, defined as therapy given between leukapheresis and the start of lymphodepleting (LD) chemotherapy were also included. Kaplan-Meier analysis was used to calculate median progression-free survival (PFS) and overall-survival (OS). Results: 112 patients were leukapheresed with intention to receive commercial CAR T as 2L (103 axi-cel, 9 liso-cel). All but two patients received CAR T infusion (one manufacturing failure and one delayed due to infection). The median follow-up of the cohort was 6.2 months (95%CI 4.32 - 7.61). Median age of the cohort was 66.5 yrs (range 23-83) with male predominance (66%). The median international prognostic index (IPI) score was 3 (range 0-5) and 72% of the patients had stage III/IV disease. Fifty-nine percent of the patients had primary refractory disease. The median time between documentation of progression after 1L therapy and patient evaluation for 2L CAR T was 22 days (IQR 15-32). The median time from patient evaluation to CAR T infusion was 47.5 days (IQR 36.25 - 67). Forty-two patients (37.5%) received disease-holding therapy and 77 (69%) received bridging therapy. Twenty-nine patients (26%), received both disease-holding therapy and bridging therapy. Receipt of these therapies was associated with longer CAR T evaluation-to-infusion times, with a median time of 61 days for patients who received both disease-holding therapy and bridging therapy, as opposed to 38 days for patients who didn't receive either therapy. This is presumably related to washout requirements and the time needed to allow for count recovery prior to either leukapheresis or LD therapy. Among 110 CAR T infused patients, cytokine release syndrome(CRS) occurred in 88% (n=97) of the patients with grade 3 or 4 in 6.3% (n=7). Immune effector cell associated neurologic syndrome (ICANS) occurred in 59% (n= 65) with grade 3 or 4 in 30% (n= 33). No deaths from CRS or ICANS were observed. Overall response rate (ORR) was 82.7% and complete remission rate was 61.8%. Median PFS and OS were not reached. Point estimates of PFS and OS at six months were 64.1% (95% CI: 52.8 - 73.4), and 84.4% (95% CI: 73.8 - 90.9), respectively ( Figure 1). Conclusions: This multicenter retrospective study provides insights into practice and outcomes of SOC CAR T therapy for 2L relapsed-refractory LBCL. In clinical practice, patients commonly receive “disease-holding therapy” to control disease while organizing CAR T-cell consultation and leukapheresis. Despite the relatively short follow-up duration, the best response and toxicity profiles were comparable to those observed in the pivotal clinical trials. Updated follow-up and subgroup analysis will be presented at the meeting. SD and JYS contributed equally.