The role of chimeric antigen receptor T cells targeting more than one antigen in the treatment of B-cell malignancies.

Several products containing chimeric antigen receptor T cells targeting CD19 (CART19) have been approved for the treatment of patients with relapsed/refractory non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukaemia (ALL). Despite very impressive response rates, a significant percentage of patients experience disease relapse and die of progressive disease. A major cause of CART19 failure is loss or downregulation of CD19 expression in tumour cells, which has prompted a myriad of novel strategies aimed at targeting more than one antigen (e.g. CD19 and CD20 or CD22). Dual targeting can the accomplished through co-administration of two separate products, co-transduction with two different vectors, bicistronic cassettes or tandem receptors. In this manuscript, we review the pros and cons of each strategy and the clinical results obtained so far.

The academic point-of-care anti-CD19 chimeric antigen receptor T-cell product varnimcabtagene autoleucel (ARI-0001 cells) shows efficacy and safety in the treatment of relapsed/refractory B-cell non-Hodgkin lymphoma.

Varnimcabtagene autoleucel (var-cel) is an academic anti-CD19 chimeric antigen receptor (CAR) product used for the treatment of non-Hodgkin lymphoma (NHL) in the CART19-BE-01 trial. Here we report updated outcomes of patients with NHL treated with var-cel. B-cell recovery was compared with patients with acute lymphoblastic leukaemia (ALL). Forty-five patients with NHL were treated. Cytokine release syndrome (any grade) occurred in 84% of patients (4% grade ≥3) and neurotoxicity in 7% (2% grade ≥3). The objective response rate was 73% at Day +100, and the 3-year duration of response was 56%. The 3-year progression-free and overall survival were 40% and 52% respectively. High lactate dehydrogenase was the only covariate with an impact on progression-free survival. The 3-year incidence of B-cell recovery was lower in patients with NHL compared to ALL (25% vs. 60%). In conclusion, in patients with NHL, the toxicity of var-cel was manageable, while B-cell recovery was significantly prolonged compared to ALL. This trial was registered as NCT03144583.

CD34+CD19-CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies.

CD19-directed immunotherapies have revolutionized the treatment of advanced B-cell acute lymphoblastic leukemia (B-ALL). Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. Patients with B-ALL successfully treated with CD19-directed T cells eventually relapse, which, coupled with the early onset of CD22 expression during B-cell development, suggests that preexisting CD34+CD22+CD19- (pre)-leukemic cells represent an "early progenitor origin-related" mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B-cell development. CD34+CD19-CD22+ cells are found in diagnostic and relapsed bone marrow samples of ∼70% of patients with B-ALL, and their frequency increases twofold in patients with B-ALL in CR after CD19 CAR T-cell therapy. The median of CD34+CD19-CD22+ cells before treatment was threefold higher in patients in whom B-ALL relapsed after CD19-directed immunotherapy (median follow-up, 24 months). Fluorescence in situ hybridization analysis in flow-sorted cell populations and xenograft modeling revealed that CD34+CD19-CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that preleukemic CD34+CD19-CD22+ progenitors underlie phenotypic escape after CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual targeting as a strategy for reducing CD19- relapses. The implementation of CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subsequent monitoring of patients with B-ALL during CD19-targeted therapy is encouraged.

Infection epidemiology in relation to different therapy phases in patients with haematological malignancies receiving CAR T-cell therapy.

BACKGROUND: We described the real-life epidemiology and causes of infections on the different therapy phases in patients undergoing chimeric antigen receptor (CAR) T-cells directed towards CD19+ or BCMA+ cells. METHODS: All consecutive patients receiving CAR T-cell therapy at our institution were prospectively followed-up. We performed various comparative analyses of all patients and subgroups with and without infections. RESULTS: Ninety-one adults mainly received CAR T-cell therapy for acute leukaemia (53%) and lymphoma (33%). We documented a total of 77 infections in 47 (52%) patients, 37 (48%) during the initial neutropenic phase and 40 (52%) during the non-neutropenic phase. Infections during the neutropenic phase were mainly due to bacterial (29, 78%): catheter infections (11 [38%] cases), endogenous source (5 [17%]), and Clostridioides difficile (5 [17%]). Patients receiving corticosteroids after CAR T-cell therapy had a higher risk of endogenous infection (100% vs. 16%; p = .006). During the non-neutropenic phase, bacterial infections remained very frequent (24, 60%), mainly with catheter source (8, 33%). Respiratory tract infections were common (17, 43%). CONCLUSIONS: Infections after CAR T-cell therapy were frequent. During the neutropenic phase, it is essential to prevent nosocomial infections and balance the use of antibiotics to lower endogenous bacteraemia and Clostridial infection rates.

Current outcomes of SARS-CoV-2 Omicron variant infection in high-risk haematological patients treated early with antivirals.

OBJECTIVES: We aimed to describe the clinical outcomes and duration of viral shedding in high-risk patients with haematological malignancies hospitalized with COVID-19 during Omicron variant predominance who received early treatment with antivirals. METHODS: We conducted a prospective observational study on high-risk haematological patients admitted in our hospital between December 2021 and March 2022. We performed detection techniques on viral subgenomic mRNAs until negative results were obtained to document active, prolonged viral replication. RESULTS: This analysis included 60 consecutive adults with high-risk haematological malignancies and COVID-19. All of these patients underwent early treatment with remdesivir. Thirty-two (53%) patients received combined antiviral strategies, with sotrovimab or hyperimmune plasma being added to remdesivir. The median length of viral replication-as measured by real-time RT-PCR and/or subgenomic RNA detection-was 20 (IQR 14-28) days. Prolonged viral replication (6 weeks after diagnosis) was documented in six (10%) patients. Only two patients had prolonged infection for more than 2 months. Overall mortality was 5%, whereas COVID-19-related mortality was 0%. CONCLUSIONS: Current outcomes of high-risk patients with haematological malignancies hospitalized with COVID-19 during Omicron variant predminance are good with the use of early antiviral strategies. Persistent viral shedding is uncommon.

Axicabtagene ciloleucel compared to tisagenlecleucel for the treatment of aggressive B-cell lymphoma.

Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) are CD19-targeted chimeric antigen receptor (CAR) T cells approved for relapsed/refractory (R/R) large B-cell lymphoma (LBCL). We performed a retrospective study to evaluate safety and efficacy of axi-cel and tisa-cel outside the setting of a clinical trial. Data from consecutive patients with R/R LBCL who underwent apheresis for axi-cel or tisa-cel were retrospectively collected from 12 Spanish centers. A total of 307 patients underwent apheresis for axi-cel (n=152) and tisa-cel (n=155) from November 2018 to August 2021, of which 261 (85%) received a CAR T infusion (88% and 82%, respectively). Median time from apheresis to infusion was 41 days for axi-cel and 52 days for tisa-cel (P=0.006). None of the baseline characteristics were significantly different between both cohorts. Both cytokine release syndrome and neurologic events (NE) were more frequent in the axi-cel group (88% vs. 73%, P=0.003, and 42% vs. 16%, P<0.001, respectively). Infections in the first 6 months post-infusion were also more common in patients treated with axi-cel (38% vs. 25%, P=0.033). Non-relapse mortality was not significantly different between the axi-cel and tisa-cel groups (7% and 4%, respectively, P=0.298). With a median follow-up of 9.2 months, median PFS and OS were 5.9 and 3 months, and 13.9 and 11.2 months for axi-cel and tisa-cel, respectively. The 12-month PFS and OS for axi-cel and tisa-cel were 41% and 33% (P=0.195), 51% and 47% (P=0.191), respectively. Factors associated with lower OS in the multivariate analysis were increased lactate dehydrogenase, ECOG ≥2 and progressive disease before lymphodepletion. Safety and efficacy results in our real-world experience were comparable with those reported in the pivotal trials. Patients treated with axi-cel experienced more toxicity but similar non-relapse mortality compared with those receiving tisa-cel. Efficacy was not significantly different between both products.

Evaluation of cell collection efficiency in non-mobilized adult donors for autologous chimeric antigen receptor T-cell manufacturing.

BACKGROUND AND OBJECTIVES: Data about collection efficiency 1 (CE1), which takes into account blood cell counts before and after collection, thus providing a more accurate estimate, in the collection of autologous T lymphocytes by apheresis for chimeric antigen receptor (CAR) T-cells remain scarce. We evaluated donor- and procedure-related characteristics that might influence the CE1 of lymphocytes. MATERIALS AND METHODS: We retrospectively reviewed all mononuclear cell (MNC) collections) performed for CAR T-cell manufacturing in our institution from May 2017 to June 2021 in adult patients. Age, gender, weight, total blood volume (TBV), prior haematopoietic cell transplant, diagnosis, days between last treatment and apheresis, pre-collection cell counts, duration of apheresis, TBV processed, vascular access, inlet flow and device type were analysed as potential factors affecting CE1 of lymphocytes. RESULTS: A total of 127 autologous MNC collections were performed on 118 patients diagnosed with acute lymphoblastic leukaemia (n = 53, 45%), non-Hodgkin lymphoma (n = 40, 34%), multiple myeloma (n = 19, 16%), and chronic lymphocytic leukaemia (n = 6, 5%). The median CE1 of lymphocytes was 47% (interquartile range: 32%-65%). In multiple regression analysis, Amicus device was associated with higher CE1 of lymphocytes (p = 0.01) and lower CE1 of platelets (p < 0.01) when compared with Optia device. CONCLUSION: The knowledge of the MNC and lymphocyte CE1 of each apheresis device used to collect cells for CAR T therapy, together with the goal of the number of cells required, is essential to define the volume to be processed and to ensure the success of the collection.

Results of ARI-0001 CART19 cell therapy in patients with relapsed/refractory CD19-positive acute lymphoblastic leukemia with isolated extramedullary disease.

We evaluated outcomes of 18 patients with isolated extramedullary disease (iEMD) relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) treated with the CD19-directed CAR T cells ARI-0001 in two centers (adult and pediatric), including patients treated in the CART19-BE-01 trial and the consecutive compassionate use program. iEMD was detected by PET-CT in 78% (14/18), and/or by cerebrospinal fluid analysis in 28% (5/18). Patients received cyclophosphamide and fludarabine followed by 1 × 106 ARI-0001 cells/kg, initially as a single dose (first patient) and later split into three fractions (10%, 30%, and 60%). Cytokine release syndrome (CRS) occurred in 50% (9/18) of patients, with no cases of grade ≥3 CRS, and 1 case (6%) of grade 1 neurotoxicity. Tocilizumab was used in 6% of patients (1/18). Procedure-related mortality was 0% at 2 years. Objective responses were seen in 94% (95% confidence interval [CI]: 73%-99%) of patients, with complete responses (CR) seen in 78% (95% CI: 52%-94%) of them. Progression-free and overall survival were 49% (95% CI: 30%-79%) and 61% (95% CI: 40%-92%) at 2 years. In conclusion, the use of ARI-0001 cells in patients with R/R ALL and iEMD was associated with a safety and efficacy profile that is comparable with what is observed in patients with marrow involvement and in line with other CART19 products.

CART19-BE-01: A Multicenter Trial of ARI-0001 Cell Therapy in Patients with CD19+ Relapsed/Refractory Malignancies.

We evaluated the administration of ARI-0001 cells (chimeric antigen receptor T cells targeting CD19) in adult and pediatric patients with relapsed/refractory CD19+ malignancies. Patients received cyclophosphamide and fludarabine followed by ARI-0001 cells at a dose of 0.4-5 × 106 ARI-0001 cells/kg, initially as a single dose and later split into 3 fractions (10%, 30%, and 60%) with full administration depending on the absence of cytokine release syndrome (CRS). 58 patients were included, of which 47 received therapy: 38 with acute lymphoblastic leukemia (ALL), 8 with non-Hodgkin's lymphoma, and 1 with chronic lymphocytic leukemia. In patients with ALL, grade ≥3 CRS was observed in 13.2% (26.7% before versus 4.3% after the amendment), grade ≥3 neurotoxicity was observed in 2.6%, and the procedure-related mortality was 7.9% at day +100, with no procedure-related deaths after the amendment. The measurable residual disease-negative complete response rate was 71.1% at day +100. Progression-free survival was 47% (95% IC 27%-67%) at 1 year: 51.3% before versus 39.5% after the amendment. Overall survival was 68.6% (95% IC 49.2%-88%) at 1 year. In conclusion, the administration of ARI-0001 cells provided safety and efficacy results that are comparable with other academic or commercially available products. This trial was registered as ClinicalTrials.gov: NCT03144583.

Cost-Effectiveness Analysis of Axicabtagene Ciloleucel vs. Standard of Care in Second-Line Treatment for Relapsed/Refractory Large B-Cell Lymphoma in Spain.

PURPOSE: To estimate the cost-effectiveness of axi-cel vs. salvage immunochemotherapy followed by high-dose chemotherapy and autologous stem-cell transplantation (HDT+ASCT) for responders to second-line treatment for relapsed/refractory (R/R) large B-cell lymphoma (LBCL). METHODS: A partitioned survival mixture-cure model comprising three health states was used to estimate the costs, life years gained (LYG), and quality-adjusted life years (QALYs) accumulated over a lifetime horizon. Overall survival, event-free survival, and time to the next treatment with axi-cel and HDT+ASCT were derived from the ZUMA-7 study. The total costs (EUR, 2022) included drug acquisition and administration, ASCT, subsequent treatment, disease and adverse event management, and palliative care. The unitary costs were derived from local databases and the literature. A 3% discount rate was applied to the costs and outcomes. RESULTS: Compared with HDT+ASCT, axi-cel provided higher LYG per patient (10.00 vs. 8.28 LYG/patient) and greater QALYs gained per patient (7.85 vs. 6.04 QALY/patient). The lifetime total costs were 343,581 EUR/patient with axi-cel vs. 257,994 EUR/patient with IQT+ASCT. The incremental cost-effectiveness ratio of axi-cel vs. HDT+ASCT was 49,627 EUR/LYG, and the incremental cost-utility ratio was 47,309 EUR/QALY. Sensitivity analyses confirmed the robustness of the model. CONCLUSION: Axi-cel is a potentially cost-effective alternative to HDT+ASCT for the treatment of R/R DLBCL in Spain.

CD19-Directed CAR T-Cells in a Patient With Refractory MOGAD: Clinical and Immunologic Follow-Up for 1 Year.

OBJECTIVES: In MOG antibody-associated disease (MOGAD), relapse prevention and the treatment approach to refractory symptoms are unknown. We report a patient with refractory MOGAD treated with CD19-directed CAR T-cells. METHODS: CD19-directed CAR T-cells (ARI-0001) were produced in-house by lentiviral transduction of autologous fresh leukapheresis and infused after a conventional lymphodepleting regimen. RESULTS: A 18-year-old man developed 2 episodes of myelitis associated with serum MOG-IgG, which were followed by 6 episodes of left optic neuritis (ON) and sustained the presence of MOG-IgG over 6 years despite multiple immunotherapies. After the sixth episode of ON, accompanied by severe residual visual deficits, CAR T-cell treatment was provided without complications. Follow-up of cell counts showed complete depletion of CD19+ B cells at day +7; reconstituted B cells at day +141 showing a naïve B-cell phenotype, and low or absent memory B cells and plasmablasts for 1 year. MOG-IgG titers have remained undetectable since CAR T-cell infusion. The patient had an early episode of left ON at day +29, when MOG-IgG was already negative, and since then he has remained free of relapses without immunotherapy for 1 year. DISCUSSION: This clinical case shows that CD19-directed CAR T-cell therapy is well-tolerated and is a potential treatment for patients with refractory MOGAD. CLASSIFICATION OF EVIDENCE: This provides Class IV evidence. It is a single observational study without controls.

Chimeric Antigen Receptor T-Cell Postinfusion Fever: Infection Profile, Clinical Parameters, and Biomarkers Trends to Assist Antibiotic Stewardship.

Background: This study aimed to describe documented infections associated with postinfusion fever after CAR T-cell therapy and to evaluate daily changes in vital signs, laboratory results, and the National Early Warning Score (NEWS) in patients with and without confirmed bacterial infections following fever onset, with the objective of assisting in antibiotic stewardship. Methods: This was a retrospective, observational study including all consecutive adult patients who received CAR T-cell therapy. Documented infection in the first fever episode after infusion, and clinical and analytic trend comparison of patients with bacterial documented infections and those without documented infections, are described. Results: Among 152 patients treated with CAR T-cell therapy, 87 (57.2%) had fever within 30 days of infusion, with a median time from infusion to fever of 3 (interquartile range, 2-5) days. Of these 87 patients, 82 (94.3%) received broad-spectrum antibiotics. Infection was documented in 9 (10.3%) patients and only 4 (4.6%) had bacterial infections. Clinical signs and biomarkers were similar in patients with bacterial documented infection and in those without documented infection at fever onset. Fever, tachycardia, and high C-reactive protein levels remained high during the first 3 days after CAR T-cell infusion, even when no infection was documented. Conclusions: Fever is a common symptom following CAR T-cell infusion and is largely treated with broad-spectrum antibiotics. However, confirmed bacterial documented infections after the first fever post-CAR T-cell infusion are very unusual. Because clinical parameters and biomarkers are not useful for identifying infectious fever, other methods should be assessed to ensure the proper use of antibiotics.

Integrative single-cell multi-omics of CD19-CARpos and CARneg T cells suggest drivers of immunotherapy response in B cell neoplasias.

The impact of phenotypic, clonal, and functional heterogeneity of chimeric antigen receptor (CAR)-T cells on clinical outcome remains understudied. Here, we integrate clonal kinetics with transcriptomic heterogeneity resolved by single-cell omics to interrogate cellular dynamics of non-transduced (CARneg) and transduced (CARpos) T cells, in the infusion product (IP) and at the CAR-T cell expansion peak in five B cell acute lymphoblastic leukemia (B-ALL) patients treated with CD19CAR-T cells (varni-cel). We identify significant differences in cellular dynamics in response to therapy. CARpos T cells at IP of complete response patients exhibit a significantly higher CD4:CD8 ratio, validated in a larger cohort B-ALL patients (n = 47). Conversely, at the expansion peak, there is a clonal expansion of CD8+ effector memory and cytotoxic T cells. Cytotoxic CARpos γδ-T cells expansion correlates with treatment efficacy validated in a cohort of B-ALL (n = 18) and diffuse large B cell lymphoma (DLBCL) patients (n = 58). Our data provide insights into the complexity of T cell responses following CAR-T cell therapy and suggest drivers of immunotherapy response.

Anti-CD19 CAR-T Cell Therapy in Elderly Patients: Multicentric Real-World Experience from GETH-TC/GELTAMO.

Chimeric antigen receptor (CAR)-T cell therapy is approved for the treatment of relapsed/refractory (R/R) large B cell lymphoma (LBCL). However, elderly patients might not be candidates for this therapy due to its toxicity, and criteria for candidate selection are lacking. Our aim was to analyze efficacy and toxicity results of CAR-T cell therapy in the population of patients 70 years and older as compared to those obtained in younger patients in the real-world setting. A multicentric retrospective study was performed including patients with R/R aggressive LBCL who received commercial CAR-T cell therapy with either tisagenlecleucel or axicabtagene ciloleucel within the Spanish Group of Hematopoietic Transplant and Cell Therapy/Spanish Group of Lymphomas and Autologous Transplant (GETH-TC/GELTAMO) centers between 2019 and 2023. As of August 2023, 442 adult patients with aggressive LBCL underwent apheresis for CAR-T cell therapy as third or subsequent line and follow-up data was collected. Of 412 infused patients, 71 (17%) were 70 years or older. Baseline characteristics, product selection, and characteristics at apheresis (including disease status, Ann Arbor stage, revised international prognosis index (R-IPI), bulky disease, lactate dehydrogenase [LDH] and ECOG [Eastern Cooperative Group performance status]) were comparable between groups. Median time from both approval to infusion and apheresis to infusion did not differ. No differences were found between groups in overall and complete response rates at 1 and 3 months. With a median follow-up of 12.2 months (range 1-44), 12-month progression-free survival (PFS) and overall survival (OS) were comparable between groups (35.2% in <70 years vs. 35.9% in ≥70 years (P = .938) and 51.1% and 52.1% (P = .885), respectively). Age ≥70 years did not affect PFS (hazard ratio (HR) 0.98, P = .941) and OS (HR 0.97, P = .890) in the univariate and multivariate analysis. Cytokine release syndrome (CRS) was observed in 82% of patients <70 years old and 84.5% in ≥ 70 years old (P = .408). Grade ≥3 CRS was more frequent in the older group (5% vs. 15%, P = .002). In the multivariate analysis, age ≥70 years was associated with an increased risk of grade ≥3 CRS (OR 3.7, P = .013). No differences were observed in terms of overall neurotoxicity (35% vs. 42%, P = .281) or grade ≥3 (12% vs. 17%, P = .33). The proportion of patients with infections, admission to the intensive care unit within the first month, and non-relapse mortality were similar between both groups. CAR-T cell therapy in patients older than 70 years showed similar efficacy to that observed in younger patients in the real-world setting. However, age ≥70 years was an independent risk factor for grades 3-4 CRS. The need for additional strategies to reduce toxicity in this population should be addressed in future studies.

Biomarkers of Efficacy and Safety of the Academic BCMA-CART ARI0002h for the Treatment of Refractory Multiple Myeloma.

PURPOSE: B-cell maturation antigen (BCMA)-chimeric antigen receptor T-cells (CART) improve results obtained with conventional therapy in the treatment of relapsed/refractory multiple myeloma. However, the high demand and expensive costs associated with CART therapy might prove unsustainable for health systems. Academic CARTs could potentially overcome these issues. Moreover, response biomarkers and resistance mechanisms need to be identified and addressed to improve efficacy and patient selection. Here, we present clinical and ancillary results of the 60 patients treated with the academic BCMA-CART, ARI0002h, in the CARTBCMA-HCB-01 trial. PATIENTS AND METHODS: We collected apheresis, final product, peripheral blood and bone marrow samples before and after infusion. We assessed BCMA, T-cell subsets, CART kinetics and antibodies, B-cell aplasia, cytokines, and measurable residual disease by next-generation flow cytometry, and correlated these to clinical outcomes. RESULTS: At cut-off date March 17, 2023, with a median follow-up of 23.1 months (95% CI, 9.2-37.1), overall response rate in the first 3 months was 95% [95% confidence interval (CI), 89.5-100]; cytokine release syndrome (CRS) was observed in 90% of patients (5% grades ≥3) and grade 1 immune effector cell-associated neurotoxicity syndrome was reported in 2 patients (3%). Median progression-free survival was 15.8 months (95% CI, 11.5-22.4). Surface BCMA was not predictive of response or survival, but soluble BCMA correlated with worse clinical outcomes and CRS severity. Activation marker HLA-DR in the apheresis was associated with longer progression-free survival and increased exhaustion markers correlated with poorer outcomes. ARI0002h kinetics and loss of B-cell aplasia were not predictive of relapse. CONCLUSIONS: Despite deep and sustained responses achieved with ARI0002h, we identified several biomarkers that correlate with poor outcomes.

Chemotherapy resistant Burkitt lymphoma: Possible novel therapies including CAR-T cell infusion.

Key Clinical Message: The prognosis of patients with relapsed or refractory (R/R) BL is poor with limited response to salvage therapy, especially for patients with early relapse (<6 months) or refractory disease. Novel therapies may be promising but need refinement. Abstract: The prognosis of patients with relapsed or refractory (R/R) BL is poor with a limited response to salvage therapy, especially for patients with early relapse (<6 months) or refractory disease. We present three cases of patients with R/R BL receiving novel therapies followed by a literature review. Amongst others, the patients received inotuzumab ozogamicin, idelalisib, ibrutinib, and CAR-T cell therapy, however, with limited response. In literature, response to several novel agents is described; however, most promising results are seen with CAR-T cell therapy. Concluding from case series, sequential CAR-T cell therapy, targeting multiple B-cell antigens, seems most promising.

Characterization of the endotheliopathy, innate-immune activation and hemostatic imbalance underlying CAR-T cell toxicities: laboratory tools for an early and differential diagnosis.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell-based immunotherapy constitutes a revolutionary advance for treatment of relapsed/refractory hematological malignancies. Nevertheless, cytokine release and immune effector cell-associated neurotoxicity syndromes are life-threatening toxicities in which the endothelium could be a pathophysiological substrate. Furthermore, differential diagnosis from sepsis, highly incident in these patients, is challenging. Suitable laboratory tools could be determinant for their appropriate management. METHODS: Sixty-two patients treated with CAR-T cell immunotherapy for hematological malignancies (n=46 with CD19-positive diseases, n=16 with multiple myeloma) were included. Plasma samples were obtained: before CAR-T cell infusion (baseline); after 24-48 hours; at suspicion of any toxicity onset and 24-48 hours after immunomodulatory treatment. Biomarkers of endothelial dysfunction (soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble TNF receptor 1 (sTNFRI), thrombomodulin (TM), soluble suppression of tumorigenesis-2 factor (ST2), angiopoietin-2 (Ang-2)), innate immunity activation (neutrophil extracellular traps (NETs), soluble C5b-9 (sC5b-9)) and hemostasis/fibrinolysis (von Willebrand Factor antigen (VWF:Ag), ADAMTS-13 (A13), α2-antiplasmin (α2-AP), plasminogen activator inhibitor-1 antigen (PAI-1 Ag)) were measured and compared with those in cohorts of patients with sepsis and healthy donors. RESULTS: Patients who developed CAR-T cell toxicities presented increased levels of sVCAM-1, sTNFRI and ST2 at the clinical onset versus postinfusion values. Twenty-four hours after infusion, ST2 levels were good predictors of any CAR-T cell toxicity, and combination of ST2, Ang-2 and NETs differentiated patients requiring intensive care unit admission from those with milder clinical presentations. Association of Ang-2, NETs, sC5b-9, VWF:Ag and PAI-1 Ag showed excellent discrimination between severe CAR-T cell toxicities and sepsis. CONCLUSIONS: This study provides relevant contributions to the current knowledge of the CAR-T cell toxicities pathophysiology. Markers of endotheliopathy, innate immunity activation and hemostatic imbalance appear as potential laboratory tools for their prediction, severity and differential diagnosis.

Lymphodepletion - an essential but undervalued part of the chimeric antigen receptor T-cell therapy cycle.

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens.

Anakinra for Refractory Cytokine Release Syndrome or Immune Effector Cell-Associated Neurotoxicity Syndrome after Chimeric Antigen Receptor T Cell Therapy.

Chimeric antigen receptor-engineered (CAR)-T cell therapy remains limited by significant toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The optimal management of severe and/or refractory CRS/ICANS remains ill-defined. Anakinra has emerged as a promising agent based on preclinical data, but its safety and efficacy in CAR-T therapy recipients are unknown. The primary objective of this study was to evaluate the safety of anakinra to treat refractory CRS and ICANS after CAR-T therapy. The secondary objective was to evaluate the impact of key treatment-, patient-, and disease-related variables on the time to CRS/ICANS resolution and treatment-related mortality (TRM). We retrospectively analyzed the outcomes of 43 patients with B cell or plasma cell malignancies treated with anakinra for refractory CRS or ICANS at 9 institutions in the United States and Spain between 2019 and 2022. Cause-specific Cox regression was used to account for competing risks. Multivariable cause-specific Cox regression was used to estimate the effect of anakinra dose on outcomes while minimizing treatment allocation bias by including age, CAR-T product, prelymphodepletion (pre-LD) ferritin, and performance status. Indications for anakinra treatment were grade ≥2 ICANS with worsening or lack of symptom improvement despite treatment with high-dose corticosteroids (n = 40) and grade ≥2 CRS with worsening symptoms despite treatment with tocilizumab (n = 3). Anakinra treatment was feasible and safe; discontinuation of therapy because of anakinra-related side effects was reported in only 3 patients (7%). The overall response rate (ORR) to CAR-T therapy was 77%. The cumulative incidence of TRM in the whole cohort was 7% (95% confidence interval [CI], 2% to 17%) at 28 days and 23% (95% CI, 11% to 38%) at 60 days after CAR-T infusion. The cumulative incidence of TRM at day 28 after initiation of anakinra therapy was 0% in the high-dose (>200 mg/day i.v.) recipient group and 47% (95% CI, 20% to 70%) in the low-dose (100 to 200 mg/day s.c. or i.v.) recipient group. The median cumulative incidence of CRS/ICANS resolution from the time of anakinra initiation was 7 days in the high-dose group and was not reached in the low-dose group, owing to the high TRM in this group. Univariate Cox modeling suggested a shorter time to CRS/ICANS resolution in the high-dose recipients (hazard ratio [HR], 2.19; 95% CI, .94 to 5.12; P = .069). In a multivariable Cox model for TRM including age, CAR-T product, pre-LD ferritin level, and pre-LD Karnofsky Performance Status (KPS), higher anakinra dose remained associated with lower TRM (HR, .41 per 1 mg/kg/day increase; 95% CI, .17 to .96; P = .039. The sole factor independently associated with time to CRS/ICANS resolution in a multivariable Cox model including age, CAR-T product, pre-LD ferritin and anakinra dose was higher pre-LD KPS (HR, 1.05 per 10% increase; 95% CI, 1.01 to 1.09; P = .02). Anakinra treatment for refractory CRS or ICANS was safe at doses up to 12 mg/kg/day i.v. We observed an ORR of 77% after CAR-T therapy despite anakinra treatment, suggesting a limited impact of anakinra on CAR-T efficacy. Higher anakinra dose may be associated with faster CRS/ICANS resolution and was independently associated with lower TRM. Prospective comparative studies are needed to confirm our findings.