CD19-directed Chimeric Antigen Receptor T-cell Therapy Research Articles

EASIX and m-EASIX predict CRS and ICANS in pediatric and AYA patients after CD19-CAR T-cell therapy

AbstractCytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) are complications of CD19-directed chimeric antigen receptor (CD19-CAR) T-cell therapy. The Endothelial Activation and Stress Index (EASIX) and modified EASIX (m-EASIX) scores have been retrospectively proven to be predictive of CRS and ICANS in adult CAR T-cell recipients. However, these scores have not been evaluated in pediatric cohorts. We retrospectively report on 76 pediatric and adolescent and young adult (AYA) patients with relapsed/refractory B-cell acute lymphoblastic leukemia treated with CD19-CAR T cells at St. Jude Children’s Research Hospital or John’s Hopkins Hospital. Data included patient, disease, and treatment characteristics. EASIX and m-EASIX scores were calculated at days –5 before, 0, and +3 after CAR T-cell infusion. CRS and ICANS occurred in 47 and 17 patients, respectively. At all evaluated time points, the median EASIX scores were higher for patients who developed severe CRS and any grade ICANS, and the median m-EASIX scores were higher in patients who developed severe CRS and severe ICANS than those with no/mild CRS/ICANS. Receiver operating characteristic curve analysis showed that both scores were strong predictors of CRS, especially severe CRS, at all time points. Any grade and severe ICANS were best predicted by both scores at day +3. m-EASIX uniformly outperformed EASIX, except for predicting any grade ICANS. Our results validate the potential utility of EASIX and m-EASIX scores for predicting CAR T-cell–related complications for pediatric and AYA patients.

CD19-directed CART therapy for T-cell/histiocyte–rich large B-cell lymphoma

AbstractT-cell/histiocyte–rich large B-cell lymphoma (THRLBCL) is a rare histologic variant of LBCL. Limited data regarding CD19–directed chimeric antigen receptor T-cell (CART) therapy in relapsed/refractory (R/R) THRLBCL suggest poor efficacy. We investigated CART outcomes for R/R THRLBCL through the Center for International Blood and Marrow Transplant Research registry. A total of 58 adult patients with R/R THRLBCL who received commercial CD19-CART therapy between 2018 and 2022 were identified. Most patients (67%) had early relapse of disease (45% primary refractory) with a median of 3 (range, 1-7) prior therapies and were treated with axicabtagene ciloleucel (69%). At median follow-up of 23 months after CART therapy, 2-year overall and progression-free survival were 42% (95% confidence interval [CI], 27-57) and 29% (95% CI, 17-43), respectively. In univariable analysis, poor performance status before CART therapy was associated with higher mortality (hazard ratio, 2.35; 95%CI, 1.02-5.5). The 2-year cumulative incidences of relapse/progression and nonrelapse mortality were 69% and 2%, respectively. Grade ≥3 cytokine release syndrome and immune effector cell–associated neurologic syndrome occurred in 7% and 15% of patients, respectively. In this largest analysis of CD19-CART therapy for R/R THRLBCL, ∼30% of patients were alive and progression free 2 years after CART therapy. Despite a high incidence of progression (69% at 2 years), these results suggest a subset of patients with R/R THRLBCL may have durable responses with CARTs.

CD22-directed CAR T-cell therapy for large B-cell lymphomas progressing after CD19-directed CAR T-cell therapy: a dose-finding phase 1 study

Outcomes are poor for patients with large B-cell lymphoma who relapse after CD19-directed chimeric antigen receptor (CAR) T-cell therapy (CAR19). CD22 is a nearly universally expressed B-cell surface antigen and the efficacy of a CD22-directed CAR T-cell therapy (CAR22) in large B-cell lymphoma is unknown, which was what we aimed to examine in this study. In this single centre, open-label, dose-escalation phase 1 trial, we intravenously administered CAR22 at two dose levels (1 million and 3 million CAR22-positive T cells per kg of bodyweight) to adult patients (aged ≥18 years) who relapsed after CAR19 or had CD19-negative large B-cell lymphoma. The primary endpoints were manufacturing feasibility, safety measured by the incidence and severity of adverse events and dose-limiting toxicities, and identification of the maximum tolerated dose (ie, the recommended phase 2 dose). This study is registered with ClinicalTrials.gov (NCT04088890) and is active, but closed for enrolment. From Oct 17, 2019, to Oct 19, 2022, a total of 41 patients were assessed for eligibility; however, one patient withdrew. 40 patients underwent leukapheresis and 38 (95%) had CAR T-cell products manufactured successfully. The median age was 65 years (range 25-84), 17 (45%) were women, 32 (84%) had elevated pretreatment lactate dehydrogenase, 11 (29%) had refractory disease to all previous therapies, and patients had received a median of four lines of previous therapy (range 3-8). Of the 38 patients treated, 37 (97%) had relapsed after previous CAR19. The identified maximum tolerated dose was 1 million CAR T cells per kg. Of 29 patients who received the maximum tolerated dose, no patients developed a dose-limiting toxicity or grade 3 or higher cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, or immune effector cell-associated haemophagocytic lymphohistiocytosis-like syndrome. This trial identifies CD22 as an immunotherapeutic target in large B-cell lymphoma and demonstrates the durable clinical activity of CAR22 in patients with disease progression after CAR19 therapy. Although these findings are promising, it is essential to recognise that this is a phase 1 dose-finding study. Further investigations are warranted to establish the long-term efficacy and to delineate the patient subgroups that will derive the most benefit from this therapeutic approach. National Cancer Institute, National Institutes of Health, Stanford Cancer Institute, Leukemia & Lymphoma Society, Parker Institute for Cancer Immunotherapy, Lymph & Co, and the European Hematology Association.

Relapsed/Refractory Diffuse Large B-Cell Lymphoma: Is There Still a Role for Autologous Stem Cell Transplantation in the CAR T-Cell Era?

Recently, CD19-directed chimeric antigen receptor (CAR) T-cell therapies have revolutionized treatment strategies for diffuse large B-cell lymphoma (DLBCL). CAR T-cell therapy is increasingly used as a second-line therapy for patients with DLBCL with early relapse or refractoriness to initial chemoimmunotherapy and displaced high-dose chemotherapy, followed by autologous stem cell transplantation (ASCT) as the standard of care for these patients. However, patients with late relapse or chemosensitive disease still benefit from autologous stem cell transplantation. We will review practice-changing studies in early relapse (ZUMA-7 and TRANSFORM) under consideration of the negative BELINDA trial, with a focus on register data, comparing CAR T-cell therapy and ASCT for patients responding to salvage therapy.

Circulating Tumor DNA as a Complementary Prognostic Biomarker during CAR-T Therapy in B-Cell Non-Hodgkin Lymphomas.

The emergence of CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment paradigm for R/R B-cell NHLs. However, challenges persist in accurately evaluating treatment response and detecting early relapse, necessitating the exploration of novel biomarkers. Circulating tumor DNA (ctDNA) via liquid biopsy is a non-invasive tool for monitoring therapy efficacy and predicting treatment outcomes in B-NHL following CAR-T therapy. By overcoming the limitations of conventional imaging modalities, ctDNA assessments offer valuable insights into response dynamics, molecular mechanisms of resistance, and early detection of molecular relapse. Integration of ctDNA monitoring into clinical practice holds promise for personalized therapeutic strategies, guiding the development of novel targeted therapies, and enhancing patient outcomes. However, standardization of assay methodologies and consensus on clinical response metrics are imperative to unlock the full potential of ctDNA in the management of B-NHL. Prospective validation of ctDNA in clinical trials is necessary to establish its role as a complementary decision aid.

Sequencing of Anti-CD19 Therapies in the Management of Diffuse Large B-Cell Lymphoma.

Several second- and third-line immunotherapeutic options for patients with relapsed or refractory diffuse large B-cell lymphoma ineligible for autologous stem cell transplant are directed against the B-cell antigen cluster of differentiation 19 (CD19). The anti-CD19 monoclonal antibody tafasitamab, paired with the immunomodulator lenalidomide, mediates antibody-dependent cellular toxicity and phagocytosis; the antibody-drug conjugate loncastuximab tesirine delivers the DNA cross-linking agent tesirine via CD19 binding and internalization; and CD19-directed chimeric antigen receptor T-cell therapy (CAR-T) products are engineered from autologous T cells. Although CD19 expression is assessed at diagnosis, clinically relevant thresholds of CD19 expression-which may not be detectable using current routine methodologies-have not been defined and may vary between CD19-directed treatment modalities. Determining optimal treatment sequencing strategies for CD19-directed therapy is hampered by the exclusion of patients who have received prior CD19-directed therapies from major clinical trials. Antigen escape, which is attributed to mechanisms including epitope loss and defective cell surface trafficking of CD19, is an important cause of CAR-T failure. Limited data suggest that CD19 expression may be maintained after non-CAR-T CD19-directed therapy, and retrospective analyses indicate that some patients with disease relapse after CAR-T may benefit from subsequent CD19-directed therapy. To date, clinical evidence on the effect of anti-CD19 therapy prior to CAR-T has been limited to small case series. Prospective studies and detailed analyses are needed to understand how pretreatment and posttreatment CD19 expression correlates with clinical responses to subsequent CD19-directed therapy to fully maximize treatment strategies.

Monocytes in leukapheresis products affect the outcome of CD19–targeted CAR T-cell therapy in patients with lymphoma

AbstractCD19–directed chimeric antigen receptor (CAR) T cells can induce durable remissions in relapsed/refractory large B-cell lymphomas (R/R LBCLs), but 60% of patients do not respond or relapse. Biological mechanisms explaining lack of response are emerging, but they are largely unsuccessful in predicting disease response at the patient level. Additionally, to maximize the cost-effectiveness of CAR T-cell therapy, biomarkers able to predict response and survival before CAR T-cell manufacturing would be desirable. We performed transcriptomic and functional evaluations of leukapheresis products in 95 patients with R/R LBCL enrolled in a prospective observational study, to identify correlates of response and survival to tisagenlecleucel and axicabtagene ciloleucel. A signature composed of 4 myeloid genes expressed by T cells isolated from leukapheresis products is able to identify patients with a very short progression-free survival (PFS), highlighting the impact of monocytes in CAR T-cell therapy response. Accordingly, response and PFS were also negatively influenced by high circulating absolute monocyte counts at the time of leukapheresis. The combined evaluation of peripheral blood monocytes at the time of leukapheresis and the 4-gene signature represents a novel tool to identify patients with R/R LBCL at very high risk of progression after CAR T-cell therapy and could be used to plan trials evaluating CAR T cells vs other novel treatments or allogeneic CAR T cells. However, it also highlights the need to incorporate monocyte depletion strategies for better CAR T production.

Transforming CLL management with immunotherapy: Investigating the potential of CAR T-cells and bispecific antibodies

Immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies or T-cell engagers, have revolutionized the treatment landscape for various B-cell malignancies, including B-acute lymphoblastic leukemia and many non-Hodgkin lymphomas. Despite their significant impact on these malignancies, their application in chronic lymphocytic leukemia (CLL) management is still largely under investigation. Although the initial success of CD19-directed CAR T-cell therapy was observed in 3 multiply relapsed CLL patients, with 2 of them surviving over 10 years without relapse, recent CAR T-cell therapy trials in CLL have shown reduced response rates compared to their efficacy in other B-cell malignancies. One of the challenges with using immunotherapy in CLL is the compromised T-cell fitness from persistent CLL-related antigenic stimulation, and an immunosuppressive tumor microenvironment (TME). These challenges underscore a critical gap in therapeutic options for CLL patients intolerant or resistant to current therapies, emphasizing the imperative role of effective immunotherapy. Encouragingly, innovative strategies are emerging to overcome these challenges. These include integrating synergistic agents like ibrutinib to enhance CAR T-cell function and persistence and engineering newer CAR T-cell constructs targeting diverse antigens or employing dual-targeting approaches. Bispecific antibodies are an exciting "off-the-shelf" prospect for these patients, with their investigation in CLL currently entering the realm of clinical trials. Additionally, the development of allogeneic CAR T-cells and natural killer (NK) cells from healthy donors presents a promising solution to address the diminished T-cell fitness observed in CLL patients. This comprehensive review delves into the latest insights regarding the role of immunotherapy in CLL, the complex landscape of resistance mechanisms, and a spectrum of innovative approaches to surmount therapeutic challenges.

Infections in children following chimeric antigen receptor T-cell therapy for B-cell acute lymphoblastic leukemia.

CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy is transforming care for pediatric patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). There are limited pediatric-specific data concerning the infection risks associated with CD19 CAR-T therapy and the adequacy of current antimicrobial prophylaxis guidelines for these patients. We describe the antimicrobial prophylaxis used and the types of infectious occurring in the first 100 days following CAR-T therapy for relapsed or refractory B-cell ALL in children and adolescents (≤18 years) at our centre. Twenty-seven patients received their first CAR-T infusion (CTI) during the study period. Almost all patients (96%) had a comprehensive Infectious Diseases review prior to CTI, which informed a personalised prophylaxis or fever/sepsis plan in six (22%). Overall, six (22%) patients had one or more infections during the study period including five (19%, 0.9 per 100 days-at-risk) from days 0-30 and three (n = 20, 15%, 0.6 per 100 days-at-risk) from days 31-100. Bacterial blood stream infections were the most common type of infection encountered during both time periods, and one patient had probable pulmonary aspergillosis. There were no infection-related deaths. Our study contributes important information on the spectrum of infections encountered in pediatric patients with B-ALL post CAR-T therapy. Overall, the burden of infectious complications post CAR-T therapy in our cohort is lower than previously reported in the literature. Results suggest that our prophylaxis recommendations are effective in this population.

Modified Endothelial Activation and Stress Index (mEASIX) Score and Immune Effector Cell Associated Haematotoxicity (ICAHT) Following Varnimcabtagene Autoleucel (IMN-003A), a CD19-Directed Chimeric Antigen Receptor T (CAR-T) Cell Therapy, in the Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (IMN-003A) is an anti-CD19 CAR-T cell immunotherapy for patients (pts) with relapsed refractory B cell malignancies (RR BCM) in first-in-India Industry Phase 2 IMAGINE study, administered in a fractionated (10%/30%/60%) infusion protocol. Cytokine Release Syndrome (CRS) and Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) are commonly associated adverse events with CAR-T cell therapy. The modified Endothelial Activation and Stress Index (mEASIX) score [lactate dehydrogenase (LDH) (U/L) × CRP (mg/dL) / platelets (PLT) (10 9 cells/L)] is a prognostic predictor of outcomes for patients in post-allogeneic transplant setting. Elevated LDH and low PLT levels have been associated with severe ICANS, and high IL-6 level is a recognized marker for CRS. Immune Effector Cell Associated Hemato-Toxicity (ICAHT) represents the most common toxicity after CAR-T cell therapy. CAR-HEMATOTOX score is used to risk stratify patients for cytopenia. This abstract reviews the mEASIX score and ICAHT for pts in the IMAGINE study, prognostic value of mEASIX for CRS, ICANS and responses, and correlation of ICAHT score with outcomes. Methods: mEASIX scores were calculated for each pt at lymphodepletion (LD), D0, D+1 and D+4. CRS and ICANS were graded as per American Society for Transplantation and Cellular Therapy (ASTCT) criteria. Pts were monitored for ICAHT post CAR-T infusion and risk stratified using CAR-HEMATOTOX score. ICAHT was graded using CTCAE v5.0. Appropriate statistical tools were used for analysis. Results: Twenty-four RR BCM pts (12 B-ALL; 12 B-NHL) were treated with var-cel in this study. Overall response rate (ORR) was 91.7% (22/24) at D+28 (B-ALL 91.7%; B-NHL 91.7%) and 80.9% (17/21) at D+90 (B-ALL 80%; B-NHL 81.8%). AESIs reported were CRS (Grade [G] 1 62.5%; G3+ 4.2%; overall 66.7%); ICANS (G1 4.2%; G3+ 0%; overall 4.2%); neutropenia (G3+ 91.7%; overall 100%); anemia (G3+ 29.2%; overall 95.8%); and thrombocytopenia (G3+ 20.8%; overall 91.7%). CRS median onset was D+5 and duration 3 days. No G3+ ICANS was reported. Treatment related mortality was 4.2% (n=1/24, CRS); 3 pts died of disease progression. The mEASIX score at LD, D0, D+1 and D+4 and its correlation with CRS, ICANS and responses is shown in Table 1. At D+1, mEASIX score <1 was predictive for responses (entire cohort, p=0.05; OR 15.0). At D+4, mEASIX score >1 was nearing statistical significance (p=0.07) for CRS (all grades; OR 6.0). However, mEASIX score at other timepoints was not predictive for CRS, ICANS or responses. The incidence of ICAHT was 100% (all grades; n=24), 100% neutropenia (G3+ 91.7%); 95.8% (n=23) anemia (G3+ 29.2%) and 91.7% (n=22) thrombocytopenia (G3+ 20.8%). The median time (range) to resolution of anemia, neutropenia and thrombocytopenia was 90 (11-270), 17.5 (1-181) and 9 (1-181) days respectively. Classical, biphasic and prolonged cytopenia pattern was seen for Neutropenia in 6, 2 and 16 pts respectively; for Anemia in 2, 0 and 21 patients respectively and for Thrombocytopenia in 4, 1 and 17 patients respectively. Overall, all patients, except two, were pancytopenic during their treatment course. Figure 1 shows the temporal course of var-cel mediated haematotoxicity (ICAHT). Two pts (8.3%) each received red cells, platelets, G-CSF and romiplostim; majority did not need supportive treatment. CAR-HEMATOTOX score was low risk in 18 pts and high risk in 6 pts. There was no statistical correlation between low risk (18 pts) and high risk (6 pts) with responses (17 pts, p=0.92) or with relapse / disease progression (9 pts, p=0.21). Conclusions: This is the first-in-India industry study evaluating mEASIX and ICAHT in patients treated with CAR-T cell therapy. mEASIX score <1 was predictive of responses at D+1 and score >1 at D+4 was mildly predictive of CRS. All patients developed ICAHT but only a small number of patients needed supportive treatment. Overall, var-cel was well tolerated with manageable cytopenia. The CAR-HEMATOTOX score was not predictive of relapse / disease progression in the IMAGINE study. mEASIX score may be used as an early predictor of CRS for permissive use of Tocilizumab before onset of severe symptoms. The ICAHT profile was heterogeneous, probably reflective of hematopoietic reserve, lines of treatment and downstream immune responses. The predictive role of mEASIX score and ICAHT profile in a real-world setting needs further study.

Phase I/II Study to Evaluate the Safety, Feasibility, and Efficacy of FP-1201 (Intravenous Interferon-Beta-1a) to Prevent Toxicities after CD19-Directed CAR T-Cell Therapy: Trial in Progress

BACKGROUND CD19 chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of patients with relapsed/refractory B-cell malignancies. Yet it remains limited by potentially life-threatening toxicities such as CRS and ICANS. The risk of CRS and ICANS restricts the use of CD19 CAR T-cell therapy to large academic centers and leads to high healthcare resource utilization. Current toxicity prevention strategies have shown limited efficacy to date (prophylactic steroids with axi-cel: CRS, 80%; ICANS, 60%; Oluwole, BJH, 2021; early intervention with brexu-cel: CRS, 89%; ICANS, 60%; Shah, The Lancet, 2021). Our group has shown that endothelial activation and dysfunction are associated with the development of CRS and ICANS, suggesting a key role of increased vascular permeability and blood-brain barrier damage in their pathogenesis (Hay, Blood, 2017). Type-I interferons (IFN-alpha and beta) are major regulators of CD73 expression by endothelial cells, an enzyme critical to the maintenance of endothelial integrity. CD73 breaks down extracellular pro-inflammatory ATP into anti-inflammatory adenosine. In addition, extracellular ATP has been shown to be a major and early driver of systemic inflammation upstream from IL-6 production (Cauwels, Cell Death Dis, 2014), which is associated with severe CRS and ICANS. Preclinical models have demonstrated that IFN-beta-1a treatment is associated with reduced capillary and blood-brain barrier permeability via upregulation of CD73 expression (Floris, J Neuroimmunol, 2002; Kraus., Ann Neurol, 2004; Niemela, Eur J Immunol, 2008). In humans, two recombinant IFN-beta-1a therapies are FDA-approved for the treatment of multiple sclerosis. IFN-beta-1a given intravenously (IV) maximizes the drug's bioavailability and its protective effects on the endothelium. In a study of patients with ARDS, IV IFN-beta-1a (FP-1201 and FP-1201-lyo) was safe and induced CD73 expression (Bellingan, Lancet Respir Med 2014). Given the known effects of IFN-beta-1a on preserving endothelial function and blood-brain barrier integrity, we hypothesize that IV IFN-beta-1a (FP-1201) may prevent CRS and ICANS following CD19 CAR T-cell therapy ( Figure 1A). Since higher rates of severe CRS and ICANS have been reported after axi-cel and brexu-cel, which contain CD28-costimulatory domains, we will restrict the inclusion criteria to patients treated with these products to analyze a population with a higher unmet need and a more homogeneous risk of CRS/ICANS. STUDY DESIGN AND METHODS Objectives and endpoints: Primary objective: To evaluate the safety and feasibility of FP-1201 in patients undergoing treatment with axi-cel or brexu-cel with two co-primary endpoints: i) to estimate the incidence of dose-limiting toxicity rates within the first 14 days following the last administration of FP-1201; ii) to study the type, frequency, and severity of adverse events according to the NCI CTCAE v5.0 from the first administration of FP-1201 and until day +28 after CAR T-cell infusion Secondary objectives: i) To decrease the incidence and severity of ICANS; ii) to decrease the incidence and severity of CRS; iii) to decrease corticosteroid usage; iv) to evaluate the effect of FP-1201 on anti-tumor efficacy Exploratory objectives: To characterize the in vivo effects of FP-1201 on endothelial function, the systemic cytokine milieu, and CAR T-cell function Key inclusion criteria: Adults ≥18 years of age with Karnofsky performance status ≥60% and B-cell non-Hodgkin lymphoma eligible for treatment with axi-cel or brexu-cel Key exclusion criteria: Known hypersensitivity to IFN-beta or major organ dysfunction FP-1201 administration: The FDA-approved IFN-beta-1a Rebif® will be formulated for IV administration (FP-1201). Participants will receive FP-1201 at one of four dose levels detailed in Figure 1B. Statistical design and sample size: We will use a Bayesian Optimal Interval Design to guide the FP-1201 dose escalation. We will plan to treat up to 24 participants. SUMMARY This is a phase I/II study of IV IFN-beta-1a in preventing CRS and ICANS following axi-cel and brexu-cel. The aim is to demonstrate safety and efficacy and to investigate the biologic mechanisms of IFN-beta-1a in preventing endothelial dysfunction.Additional preclinical and preliminary clinical data will be presented at the meeting. This study is registered on clinicaltrials.gov as NCT05936229.

CD19 Intron Retention Is Mechanism of CAR-T Treatment Resistance in Non-Hodgkin Lymphoma

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has become a viable treatment option for patients (pts) with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Transcriptomic markers for CD19 directed CAR-T failure have been characterized in pre-treatment B-cell acute lymphoblastic leukemia (B-ALL) but not in DLBCL. Here, we report the identification of RNA alternative splicing (AS) events associated with CD19 directed CAR-T response in pre-treatment DLBCL pts and of RNA binding proteins (RNABP) regulating these AS events. Methods: To identify putative AS markers for CAR-T treatment outcome, we performed differential AS on bulk RNA-seq samples of pre-treatment tumor biopsies in pts that had a durable response (DR), defined via remission up to 9 months post-treatment, and pts that had a non-durable response (NDR) with rMATS-turbo (Shen et al. PNAS. 2014). Intron retention (IR) events within CD19 were quantified with a secondary, more robust algorithm to ensure reproducibility and accuracy. Survival analyses were done using the survival and survminer libraries in R. Validation assays for the downstream effects CD19 IR were conducted in EJ1, Jeko-1, OCI-Ly3, SU-DHL-6, and Toledo cell lines. The surface protein expression of CD19 was assayed via flow cytometry (FC) followed by Quantibrite PE (BD Biosciences, San Jose, CA) quantification. Total CD19 protein expression was measured via western blot (WB) in the aforementioned cell lines. Analyses of CD19 IR was done via RT-PCR followed by electrophoresis, and bands were quantified with iBright Analysis Software (Thermo Fisher Scientific, Carlsbad, CA). The effect of RNABP was assessed via the creation of CRISPR/Cas9 knockout (KO) cell lines followed by WB of the target gene and quantification of CD19 surface protein expression by FC. Results: Introns 2 and 6 of CD19 are more highly expressed in NDR pts. A combined metric estimating the proportion of normal CD19 isoforms that do not express introns 2 or 6 (normal CD19) was created and DR pts expressed normal CD19 isoforms significantly higher than NDR pts (Figure 1). Normal CD19 isoforms classification performance was assessed via leave one out cross validation and achieved an overall accuracy of 67.6%. Sensitivity (classifying NDR) was 61.1% and specificity (classifying DR) was 81.3%. The expression of normal CD19 relative to the median level within the cohort was significantly associated with progress free survival (Figure 2). The expression of CD19 intron 6 was negatively correlated with CD19 surface protein expression in DLBCL cell lines. Cell lines were sorted by cells that exhibited high CD19 surface expression (bright) and low expression (dim). Cell lines sorted into the dim category expressed higher levels of intron 6 than bright cells. The correlation of RNABP expression with CD19 intron 6 expression was done in this patient cohort and the NCICCR-DLBCL cohort. Six RNABP were significantly correlated in both cohorts (r 2 > 0.2 and p-value <0.01). NOP2, one of the RNABP that was positively correlated with intron 6 expression, was knocked out in Jeko-1 cell lines to validate the putative correlation with CD19 protein expression. The resulting knock out cells exhibited lower levels of intron 6 retention and higher CD19 protein expression by FC and WB than non-transformed cells. Conclusion: Pre-treatment tumors of R/R DLBCL patients who relapse before 9 months following CD19 directed CAR-T therapy have higher expression of CD19 isoforms that retain introns 2 and 6. The upregulated expression of the CD19 isoforms retaining these introns is correlated with CD19 surface protein expression and is a marker for sustained remission following CAR-T treatment. NOP2 is correlated with CD19 intron retention and surface protein expression and could be a target for modulating CD19 expression in conjunction with CAR-T treatment. Figure 1: The expression of normal CD19 isoforms in pre-treatment tumor samples of DR (red) and NDR patients (blue). ΔPSI denotes the difference in medians between the two groups. P-value yielded by a Wilcoxon test between the two groups is reported. Figure 2: Progress free survival (PFS) stratified by expression of normal CD19 isoforms. Red - above cohort median expression of normal CD19 isoforms. Blue - below cohort median expression of normal CD19 isoforms. P-value yielded via log-rank test.

Development of Cytokine Release Syndrome Is Associated with Superior Progression-Free Survival in DLBCL Patients Receiving Axi-Cel or Liso-Cel

Introduction: Chimeric antigen receptor T-cell therapy (CAR-T) targeting CD19 is a new standard of care for diffuse large B-cell lymphoma (DLBCL) patients with primary refractory disease, early relapse after chemoimmunotherapy, or multiply relapsed disease. Three CD19-directed CAR-T products, axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), and lisocabtagene maraleucel (liso-cel), are FDA approved for relapsed/refractory (R/R) DLBCL and may cure approximately 30-40% of patients. Cytokine release syndrome (CRS), a systemic inflammatory response associated with CAR-T expansion, is a common early toxicity occurring in 42-93% of DLBCL patients in the pivotal phase 2 trials. We hypothesized that development of CRS may be associated with post-CAR-T efficacy outcomes. Methods: We retrospectively evaluated post-CAR-T outcomes for 57 consecutive patients with R/R DLBCL who received axi-cel or liso-cel infusion at the University of California San Francisco between June 2018 and December 2022. Early post-CAR-T toxicities including CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) were recorded using ASTCT consensus grading. Post-CAR-T efficacy endpoints including overall response rate (ORR), complete remission (CR) rate (best response), progression-free survival (PFS), and overall survival (OS) were evaluated for the entire cohort and compared between patients who developed any grade CRS or no CRS after CAR-T infusion. Of note, patients with high-grade B-cell lymphoma or who received tisa-cel were excluded from this study to eliminate potential confounding given poorer outcomes in these subgroups and imbalance in their relative proportions between the CRS and no CRS cohorts. Results: For the entire cohort, the median age was 63 (range 26-81) and 59% of patients were male. Diagnoses included de novo DLBCL (65%), transformed follicular lymphoma (30%), and primary mediastinal B-cell lymphoma (5%). Patients had a median of 2.5 systemic therapies (range 1-8) prior to CAR-T infusion. CAR-T products included axi-cel (70%) or liso-cel (30%). 82% of patients received fludarabine and cyclophosphamide lymphodepletion, and the remaining 18% received bendamustine (during the national fludarabine shortage). Forty-one patients (72%) experienced CRS (grade 1: 35%, grade 2: 37%), and 16 patients (28%) never developed CRS. Seventeen patients (30%) developed ICANS (grade 2: 12%, grade 3: 16%, grade 4: 2%), which occurred exclusively among patients in the CRS cohort. 93% of patients in the CRS cohort received at least one dose of tocilizumab. Baseline characteristics were well balanced between the CRS and no CRS groups with no significant differences in age, sex, performance status, DLBCL subtype, number of prior therapies, CAR-T product administered, lymphodepletion administered, or lactate dehydrogenase at the time of infusion. For the entire cohort, the ORR and CR rate were 86% and 70%, respectively. With a median follow-up of 1.1 years (range 0.2-4.5 years), the 1-year PFS and OS estimates were 61% and 91%, respectively. Median PFS was 1.7 years and median OS was not reached. Outcomes were similar for patients receiving axi-cel and liso-cel (1-year PFS 62% vs 60%, p=0.94). For patients in the CRS cohort vs no CRS cohort, the ORR was 90% vs 75% (p=0.14) and CR rate was 73% vs 63%, respectively (p=0.44). Patients in the CRS cohort had superior PFS compared to patients with no CRS (1-year PFS 70% vs 38%, HR 0.36, 95% CI 0.14-0.98, logrank p=0.011), but there was no significant difference in OS ( Figure 1). There was no significant difference in PFS between patients with grade 1 vs grade 2 CRS (1-year PFS 64% vs 75%, p=0.46). Conclusions: Although limited in sample size, our study suggests that development of CRS may be associated with more favorable post-CAR-T efficacy including superior PFS compared to patients with no CRS after axi-cel or liso-cel infusion. The more favorable outcomes in patients experiencing CRS may reflect better CAR-T proliferation and cytotoxicity compared to patients with no CRS. These findings suggest a relationship between early post-CAR-T toxicity and efficacy outcomes but require validation in larger datasets.

Manufacturing of a Subsequent Autologous CAR-T Product after Prior CAR-T Is Safe and Feasible

Introduction: Patients with large B-cell lymphoma frequently fail to achieve a durable remission following CD19-directed chimeric antigen receptor T-cell therapy (CAR-T). Unfortunately, the outcomes for patients who progress after CAR-T are dismal with the median overall survival of approximately 6 months. Trials are now demonstrating the possibility of a subsequent autologous CAR-T for treating these patients. We demonstrated a CD22-directed CAR-T (CAR22) achieved a 68% and 53% overall (ORR) and complete response (CR) rate in 38 subjects who progressed after a CD19 CAR-T (CAR19) (NCT04088890). Of the 20 patients who achieved CR, 17 never relapsed after a median follow up of 22.8 months. The safety and feasibility of manufacturing a second (or more) CAR-T from patients has largely not been addressed and some concerns have been raised regarding the theoretical risk of malignant transformation due to the repeatedly introduction of new genomic material through serial CAR-T manufacturing. Here we address the impact of residual CAR19 on CAR22 production and patient outcomes. Method: We evaluated the patients who progressed after CAR19 and underwent manufacturing for CAR22. Single-cell RNA-sequencing (scRNAseq; 10X) was used to quantify the frequency of axi-cel transcripts in CAR22 products. Patients' peripheral blood mononuclear cells (PBMCs) were collected on day 7, 14, 21, and 28 after CAR22 infusion. The FMC63 and CD22.BB.z sequence (with albumin as a control) were quantified using quantitative polymerase chain reaction (qPCR) to determine the presence of CAR19 and CAR22 within the PBMCs. The assay's limit of detection and quantification (LOQ) were calculated as 1.68 and 5.1 copies per 50ng of DNA reaction with 95% confidence, respectively. Results: Among 40 patients underwent manufacturing for CAR22, 2 failed manufacturing due to insufficient T cells in the apheresis material. One of the two patients received prior alemtuzumab in combination with a CAR19 product, likely contributing to the failure. Among the 38 who were successfully manufactured, 37 patients had received prior CAR19, including axi-cel (n = 28), tisa-cel (n = 3), liso-cel (n = 3), autologous CAR19 in clinical trial (n = 1), bispecific CD19/CD22 CAR-T (n = 1), and bispecific CD19/CD20 CAR-T (n = 1). The median progression-free survival (PFS) from the previous CAR19 infusion was 3.3 months (range from 1.0 to 40.4 months), with a median age of 64.4 years old (ranging from 24.9 to 83.9 years old) at the time of relapse. The median time from CAR19 infusion to leukapheresis for CAR22 manufacturing was 6.8 months (ranging from 1.2 to 42.9 months). Thirteen CAR22 products were available for scRNAseq; all from patients who had received axi-cel previously. The median frequency of CAR19 positive cells was approximately 68 (ranging from 0 to 2146) per million cells per CAR22 product. Residual CAR19 cell proportions were not statistically significantly different between patients who underwent CAR22 manufacturing within or after 6 months of CAR19 infusion. Thirty-one patients had available PBMCs post-CAR22 infusion for analysis. Twelve patients (39%) exhibited an increase in CAR19 qPCR transgene levels above the LOQ during CAR22 treatment. Most of these patients (n = 8) underwent leukapheresis for CAR22 manufacturing within 6 months after CAR19 infusion. The median CAR19 qPCR transgene levels increased by 1.2 log (ranging from 0.2 to 2.1 log; equivalent to 1.4 to 133.7 copies per 50ng of DNA), while CAR22 transgene levels increased by a median of 3.7 log (ranging from 0.7 to 3.9 log; equivalent to 4.7 to 9027.1 copies per 50ng DNA). In the comparison between patients who underwent leukapheresis within or beyond 6 months after CAR19 infusion, there were no statistically significant differences in the maximal CAR19 and CAR22 qPCR transgene levels. The ORR, cytokine release syndrome severity, immune effector cell-associated neurotoxicity syndrome severity and PFS after CAR22 were not statistically significantly different between patients with detectable CAR19 qPCR or not. Conclusion: We found that CAR19 was detectable in the majority of CAR22 products, but the levels of CAR19 transcripts were rare. While CAR19 cells were detected post-CAR22 infusion, the levels were frequently just at the LOQ and did not appear to contribute to toxicity or clinical outcomes. Overall, we find that serial manufacturing of autologous CAR-T products is safe and feasible.

Loss of p53 Impairs Death Receptor Expression and Confers Resistance to CD19-Directed CAR T-Cell Therapy in B-Cell Precursor Acute Lymphoblastic Leukemia

Background In recent years, new therapy strategies such as the antibody-drug conjugate inotuzumab ozogamicin, the bi-specific T-cell engager blinatumomab, and chimeric antigen receptor (CAR) T-cells have been introduced to salvage relapsed precursor B acute lymphoblastic leukemia (ALL). However, recent evidence indicates that these new therapies are ineffective in TP53-aberrant ALL, although the mechanism remains unclear. TP53 is the most frequently mutated gene in cancer, but in ALL, mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse, TP53 aberrations are found in approximately 12% of the patients and predict a very poor outcome. Consequently, relapsed TP53 deleted ALL is now classified as ‘very high risk’. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance to classical chemotherapeutics. Hence, there is a clear clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wild type and knockout models in TP53 WT ALL cell lines Nalm6 and RCH-ACV. Additionally, we prepared CD19-directed CAR T-cells with a 4-1BB co-stimulatory domain from three healthy donors by isolating, transducing, and expanding CD4+ and CD8+ T-cells separately. With flow cytometry, we assessed survival of p53 WT and p53 KO leukemic cells in the presence of CD4+ and CD8+ CAR T-cells at a 1:1 CD4:CD8 ratio. Moreover, we performed mRNA profiling by qRT-PCR and cell membrane profiling by flow cytometry to determine how loss of p53 affects CAR T mediated killing. Initial findings in cell lines were then validated in patient-derived xenografts. Results We observed that loss of p53 led to up to a 35% decrease in killing by overnight CAR T treatment in both cell line models (Figure 1A). Although loss of p53 has been linked to reduced cell surface expression of CD19 (Pan et al., 2020, Leukemia), no changes in CD19 expression were observed in response to p53 loss. In search for an explanation for the diminished killing, we performed mRNA profiling and found a reduced expression of death receptors CD95/Fas and DR5/TRAILR2 in p53-deficient cells, which was confirmed at the protein level with flow cytometry (Figure 1B). Indeed, CRISPR/Cas9 mediated loss of CD95/Fas expression in p53 wild type cells reduced CAR T mediated cell killing, consistent with a key role for CD95/Fas in T-cell mediated killing. Conversely, treatment of p53 proficient cells with MDM2 inhibitors induced expression of CD95/Fas and DR5/TRAILR2 (Figure 1B), which translated into enhanced killing of target cells. Most of these observations have already been recapitulated in a diagnosis-relapse pair of patient-derived cells in which the diagnosis was p53wild typeand the relapse was p53 deficient (Figure 1A-B). We observed lower baseline mRNA expression of CD95/Fas and DR5/TRAILR2 in p53-deficient cells, accompanied by a 50% reduction in killing relative to the p53 wild type controls. Conversely, MDM2 inhibition increased death receptor expression only in p53-proficient cells. The ongoing experiments validating enhanced killing after MDM2 inhibition in patient-derived cells will be presented during the meeting. Conclusions Loss of p53 impairs expression of CD95/Fas and DR5/TRAILR2, thereby protecting ALL cells from CAR T induced cell death. In addition, our results indicate that the p53-controlled expression of these receptors may be exploited to increase the efficacy of CAR T-cell therapy in p53-proficient patients.

Prevalence of Non-Hodgkin Lymphoma Patients at High Risk of Failure after CAR T-Cell Therapy Eligible for Bridging Radiation Therapy.

CD19-directed chimeric antigen receptor T-cell therapy (CART) has led to remarkable outcomes in patients with relapsed or refractory (R/R) non-Hodgkin's lymphomas (NHL), yet ∼50% of patients relapse. Predictive factors associated with high risk of relapse following CART have been identified in the literature. Studies have shown a potential improvement in outcomes with the use of bridging radiation (RT) prior to CART. The purpose of this study is to determine the incidence of patients meeting high risk criteria who have disease that could be encompassed in a standard radiation plan prior to CART. This was a single-center, retrospective study of patients with R/R NHL who received CART from 2018 to 2022. Data were obtained from the EHR and manual chart review was used to identify patients with disease encompassable in a standard radiation plan defined as <5 malignant lesions on pre-apheresis radiological studies. These patients were then screened for presence of ≥1 high risk criteria: bulky disease ≥10 cm, ≥1 extranodal (EN) sites, LDH ≥normal, or ≥1 lesion with SUVmax ≥10. Descriptive statistics were used to make inferences about the population. Eighty-one R/R NHL patients were evaluated with a median age of 65 years (range 23-84), 60% male, and received a median of 2 (range 1-7) prior lines of therapy. CART products included axi-cel (62%), brexu-cel (7%), liso-cel (16%), and tisa-cel (15%). Forty (49%) patients would have been eligible for bridging RT (<5 sites on pre-apheresis radiologic studies), including 29 patients who met high risk criteria: 19 had ≥1 lesion with SUVmax ≥10, 16 with bulky disease, 6 with ≥1 EN site, and 3 with elevated LDH. At 3 months post-CART, clinical response data were available for 19 high risk patients with ORR of 79% (11 CR, 4 PR, 1 SD, and 3 PD), compared to 8 patients with standard risk disease with ORR of 88% (7 CR and 1 PD). Of those with less than a CR, 8/9 met high risk criteria: 4 with bulky disease, 6 had ≥1 lesion with SUVmax ≥10, and 2 with ≥1 EN site. There were 9/40 patients who received bridging therapy, and only 2 high risk patients received bridging RT. One patient with transformed DLBCL had persistent disease in the nasopharynx following RCHOP x3C and R-HyperCVAD, and received 20 Gy/10 fx as a bridge to CART resulting in a continued CR at 23 months of follow-up. Another patient with non-DH, GCB-like DLBCL received R-CHOP x6C (achieved CR) but relapsed <1 year later and then received R-ICE x2C with PD in the left shoulder, which was radiated with 30 Gy/10 fx for pain as a bridge to CART, but experienced disease relapse 6 months after CART. Approximately 35% (28/81) of patients were classified as high risk of relapse after CART and had disease encompassable in a standard radiation plan. Of these patients, 44% (8/18) experienced either PR, SD or PD at 3 months post-CART. Future studies are needed to determine the role of bridging RT prior to CART as a strategy to improve outcomes and prevent relapse in high-risk NHL patients.

Long-Term Survivors after Failure of Chimeric Antigen Receptor T Cell Therapy for Large B Cell Lymphoma: A Role for Allogeneic Hematopoietic Cell Transplantation? A German Lymphoma Alliance and German Registry for Stem Cell Transplantation Analysis

The outcome of patients with large B cell lymphoma (LBCL) who relapse or progress after CD19-directed chimeric antigen receptor T cell therapy (CAR-T) administered as salvage therapy beyond the second treatment line is poor. However, a minority of patients become long-term survivors despite CAR-T failure. The German Lymphoma Alliance (GLA) has proposed a hierarchical management algorithm for CAR-T failure in LBCL, aimed at allogeneic hematopoietic cell transplantation (alloHCT) as definite therapy in eligible patients. The purpose of this study was to investigate characteristics, relapse patterns, and management strategies in long-term survivors after CAR-T failure, with a particular focus on the feasibility and outcome of alloHCT. This was a retrospective analysis of all evaluable patients with a relapse/progression event (REL) observed in a previously reported GLA sample between November 2018 and May 2021. REL occurred in 214 of 356 patients (60%) who underwent CAR-T for LBCL in the previous GLA study. An evaluable dataset was available for 143 of these 214 patients (67%). Twenty-six of 143 patients (18%) survived 12 months or longer from REL, 109 (76%) died within the first year after REL, and 8 (6%) were alive but had not reached the 12-month landmark. Long-term survivors had more favorable pre-CAR-T features, had a longer interval between CAR-T and REL, and had more often received a tumor biopsy after CAR-T failure, whereas the choice of the first salvage regimen had no impact. AlloHCT was feasible in 40 of 53 patients (75%) intended and resulted in a 12-month post-transplantation overall survival of 36% in those patients who underwent transplantation with sensitive or untreated REL. AlloHCT after CAR-T failure in LBCL is feasible and may be an important contributor to long-term survival, although selection bias must be taken into account. Thus, alloHCT should be considered as a reasonable treatment option for eligible patients in this setting. However, because the overall outlook after CAR-T failure remains poor, novel effective therapeutic approaches are needed, either to allow long-term disease control per se or to improve the preconditions for successful alloHCT.

CAR-T in the Treatment of Acute Myeloid Leukemia: Barriers and How to Overcome Them.

Conventional therapies for acute myeloid leukemia (AML) are characterized by high rates of relapse, severe toxicities, and poor overall survival rates. Thus, the development of new therapeutic strategies is crucial for improving the survival and quality of life of AML patients. CD19-directed chimeric antigen receptor (CAR) T-cell immunotherapy has been extremely successful in the treatment of B-cell acute lymphoid leukemia and several mature B-cell lymphomas. However, the use of CAR T-cell therapy for AML is currently prevented due to the lack of a myeloid equivalent to CD19, as currently known cell surface targets on leukemic blasts are also expressed on healthy hematopoietic stem and progenitor cells as well as their progeny. In addition, the immunosuppressive tumor microenvironment has a dampening effect on the antitumor activity of CAR-T cells. Here, we review the therapeutic challenges limiting the use of CAR T-cell therapy for AML and discuss promising novel strategies to overcome them.

Axicabtagene Ciloleucel in Combination with the 4-1BB Agonist Utomilumab in Patients with Relapsed/Refractory Large B-Cell Lymphoma: Phase 1 Results from ZUMA-11.

Chimeric antigen receptor (CAR) T-cell therapies have shown clinical benefit for patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL), yet approximately 60% of patients do not respond or eventually relapse. We investigated the safety and feasibility of the CD19-directed CAR T-cell therapy axicabtagene ciloleucel (axi-cel) in combination with the 4-1BB agonist antibody utomilumab as an approach to improve efficacy of CAR T-cell therapy. In phase 1 of the single-arm ZUMA-11 trial, patients with R/R LBCL received a single axi-cel infusion (target dose, 2 × 106 cells/kg) plus utomilumab 10 to 200 mg intravenously every 4 weeks for up to 6 months in a dose-escalation design. The primary endpoint was incidence of dose-limiting toxicities (DLT) with utomilumab. Key secondary endpoints were safety, antitumor activity, pharmacokinetics, and pharmacodynamics. No DLTs were observed among patients treated with axi-cel and utomilumab (n = 12). Grade ≥3 adverse events occurred in 10 patients (83%); none were Grade ≥3 cytokine release syndrome or neurologic events. The objective response rate was 75% and seven patients (58%) had a complete response. Peak CAR T-cell levels increased in a utomilumab dose-dependent manner up to 100 mg. Patients who received utomilumab 100 mg had persistently increased CAR T cells on days 57 to 168 compared with other dose levels. Utomilumab was associated with dose-dependent increases in IL2, IFNγ, and IL10. Utomilumab-mediated 4-1BB agonism combined with axi-cel therapy had a manageable safety profile. Dual 4-1BB and CD28 costimulation is a feasible therapeutic approach that may enhance CAR T-cell expansion in patients with LBCL.

Cellular therapy in lymphoma.

CD19-directed chimeric antigen receptor (CAR) T-cell therapy has had a dramatic impact on the natural history and survival of patients with high-risk B-cell non-Hodgkin lymphoma. Accompanying this success has been the development of new fields of medicine and investigation into toxicity risks and mitigation therapies, mechanisms of resistance and the development of novel and next generation products and strategies in order to address relapse, and issues related to global access and health care economics. This article is a survey of each of these areas as it pertains to the rapidly evolving field of CAR T-cell therapy, written by an International community of lymphoma experts, who also happen to be women.