CD19 Chimeric Antigen Receptor T-cell Therapy Research Articles

Aggressive Lymphoma after CD19 CAR T-Cell Therapy

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

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

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

Chimeric Antigen Receptor T Cells Targeting CD19 and GCC in Metastatic Colorectal Cancer

Chimeric antigen receptor (CAR) T-cell therapy (CART) has transformed the treatment landscape of hematologic cancer, but has negligible effects for adult solid cancers. In this trial, an autologous CAR T-cell product demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer (mCRC). To evaluate the safety and efficacy of guanylate cyclase-C (GCC19) CART in participants with metastatic colorectal cancer (mCRC). This single-arm, nonrandomized, phase 1 trial was conducted at the First Hospital of Jilin University from December 3, 2020, to April 13, 2022. Data analysis was conducted from May 2022 to April 2024. Adults with relapsed and refractory mCRC expressing GCC were treated with GCC19CART, a mixture of autologous CAR T cells transduced with lentiviral vectors expressing genes that encode either CD-19 CAR or GCC CAR. Safety and tolerability of CAR T-cell therapy targeting GCC in patients with mCRC without therapeutic options is capable of conferring a reasonable likeliness of clinical benefit. Other outcomes included objective response rate, progression-free survival, overall survival, and immune activation. Of 15 patients 9 (60%) were women, and the median (range) age was 44 (33-61) years. Treatment with GCC19CART was associated with the development of cytokine release syndrome and diarrhea in most patients, all of which were self-limited and manageable. The objective response rate was 40%, with a partial response in 2 of 8 and 4 of 7 patients treated with either 1 × 106 cells/kg or 2 × 106 cells/kg. Median overall survival was 22.8 months (95% CI, 13.4-26.1) at data cutoff; the median progress-free survival was 6.0 months in the high dose level group (95% CI, 3.0 to not available). The results of this nonrandomized clinical trial suggest that GCC19CART was safe and tolerable in heavily pretreated patients with mCRC and is the first CAR T-cell therapy known to produce objective clinical activity in refractory cancer. Given the paucity of effective therapeutics developed for colorectal cancer in recent decades, the observation that CD-19 CART target engagement can robustly induce GCC19CART target engagement sufficient to produce objective activity may serve as a foundation to develop effective cellular therapy in mCRC and other solid cancers. Chinese Clinical Trial Registry: ChiCTR2000040645.

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

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

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

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

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

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

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

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

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

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

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

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

Preclinical evaluation of cyclophosphamide and fludarabine combined with CD19 CAR-T in the treatment of B-cell hematologic malignancies in vivo.

Chimeric antigen receptor T (CAR-T) cell therapy has achieved marked therapeutic success in ameliorating hematological malignancies. However, there is an extant void in the clinical guidelines concerning the most effective chemotherapy regimen prior to chimeric antigen receptor T (CAR-T) cell therapy, as well as the optimal timing for CAR-T cell infusion post-chemotherapy. We employed cell-derived tumor xenograft (CDX) murine models to delineate the optimal pre-conditioning chemotherapy regimen and timing for CAR-T cell treatment. Furthermore, transcriptome sequencing was implemented to identify the therapeutic targets and elucidate the underlying mechanisms governing the treatment regimen. Our preclinical in vivo evaluation determined that a combination of cyclophosphamide and fludarabine, followed by the infusion of CD19 CAR-T cells five days subsequent to the chemotherapy, exerts the most efficacious therapeutic effect in B-cell hematological malignancies. Concurrently, RNA-seq data indicated that the therapeutic efficacy predominantly perturbs tumor cell metabolism, primarily through the inhibition of key mitochondrial targets, such as C-Jun Kinase enzyme (C-JUN). In summary, the present study offers critical clinical guidance and serves as an authoritative reference for the deployment of CD19 CAR-T cell therapy in the treatment of B-cell hematological malignancies.

Distinct Temporal Dynamic Serum Metabolite Profiles Characterize Treatment Response and Early Toxicity after B-Cell Lymphoma Directed CAR-T-Cell Therapy

Introduction: CD19 chimeric antigen receptor T-cell therapy (CD19.CAR-T) has profoundly improved treatment options for patients with relapsed/refractory B-cell lymphomas (r/r BCL), but is also associated with distinct toxicities. To elucidate host-intrinsic immunometabolic factors contributing to clinical benefit or immunotoxicity, we recently demonstrated that an increase of metabolically high-risk adipose tissue mass is associated with an elevated cytokine release syndrome (CRS) risk but also with improved survival (Haematologica 2022, CIR 2023). To gain further insights into the underlying immunometabolic mechanisms, we analyzed temporal dynamic changes of serum metabolites in r/r BCL patients during the first 14 days of CD19.CAR-T treatment. Methods: In this single-center analysis, we studied patients receiving CD19.CAR-T for r/r large BCL or mantle cell lymphoma (MCL) between 01/2019 and 12/2021 in a real-world setting. All patients were consecutively treated in our department (LMU University Hospital) and serum was prospectively collected in the morning hours from fasted patients beginning at the day of CAR-T infusion (D0), and 3-5 and 14 days later. Using liquid chromatography mass-spectrometry, we quantitatively analyzed a total of 305 water-soluble metabolites and 734 lipids. Metabolomic data was integrated with clinical patient records including CRS, 1-year progression-free survial (PFS) and amount of visceral adipose tissue (VAT). Results: In total, we measured 170 serum samples from 55 r/r LBCL and 4 r/r MCL patients, of which 52 had serial samples at all three time points (D0: 55, D3-5: 59, D14: 56). The following CAR-T products were administered: axi-cel (n = 26), tisa-cel (n = 29) and brexu-cel (n = 4). 25 (42.4%) patients developed a CRS grade ≥ 2, while 34 patients had a CRS grade 0-1. The best 90-day overall response rate was 52.5%, with a complete remission rate of 42.4%. At 1 year, 67.8% of patients progressed. From the 1039 metabolites measured, 525 metabolites passed quality control. Metabolite values were normalized and auto-scaled for further analysis. A partial least square differential analysis based on the collected time points led to a clear separation of samples with uric acid, desoxy-ribose-5-phosphate (dRibose-P), essential amino acids and (phospho)lipids having the strongest impact. Whereas 56 metabolites showed a significant increase from D0 to D14 led by uric and essential amino acids, 34 metabolites decreased over the course of treatment with the strongest reduction in dRibose-P and sphingosine phosphate. Performing a metabolic pathway enrichment analysis based on the most altered metabolite levels, we detected 22 significantly affected metabolic pathways and programs including aerobic glycolysis, urea cycle, amino acid and lipid metabolism (FDR &amp;lt; 0.1, Fig. 1A). To further delineate metabolite changes in regards to CRS, 1-year PFS and amount of VAT, we conducted a multivariate empirical bayes analysis of variance to specifically detect metabolites with differential temporal dynamics and considered all metabolites with an FDR &amp;lt; 0.1. Patients who developed a CRS ≥ 2° showed increased levels of kynurenine, phosphatidylethanolamides and lysophosphatidylserines, and decreased levels of ornithine and phosphatidylinositols. 1-year progressors showed a significant increase of several phosphatidylinositols beginning at D3-5 after CAR-T infusion, and decreased levels of phosphatidylcholines. For VAT, we used a threshold of 144 cm² which was derived from previously conducted computer tomography segmentation analyses. As expected, VAT cohorts mostly differed in fatty acid metabolism-associated metabolites including (acyl)carnitines, cholesterol esters, triglycerides and xanthine. In order to identify metabolites being associated with multiple outcomes, we looked for mutual metabolites among distinct cohort profiles. Here, one example is phosphatidylinositol-(22:5/16:0), which was increased in CRS 0-1° and 1y-progressors ( Fig. 1B). Conclusions: Our study highlights that CD19.CAR-T treatment is accompanied by broad changes in the serum metabolome of r/r BCL patients, which can be used to profile patients being affected by CAR-T-related immunotoxicity and their response to treatment. In ongoing analyses, we evaluate the use of these profiles for predictive models, and assess their impact on CAR-T cell function in-vitro.

Characteristics of Central Nervous System Relapse after CD19 CAR-T Cell Therapy in R/R NHL

Background CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable antitumor activity against relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL). However, NHL with central nervous system (CNS) relapse carries a poor prognosis and high mortality. We reported an analysis of 80 patients with NHL who underwent CAR-T cell therapy, and described the characteristics including the international prognostic index (IPI) and other clinical variables to evaluate the outcomes of CNS relapse in NHL patients treated with CAR-T cell therapy. Methods A total of 80 patients with NHL treated with CD19 CAR-T cell therapy in our center between April 2017 and November 2022 was retrospectively analyzed. Patient characteristics, laboratory parameters, and clinical outcomes were collected. CNS relapse was diagnosed by cerebrospinal fluid cytology, MRI or PET-CT. Results Baseline characteristics of all 80 patients are summarized in Table 1. The median age was 58.5 (range, 10 to 79) years, and 57.5% of patients were male. Overall, 67 (83.8%) patients achieved complete remission (CR) or partial remission (PR) after CD19 CAR-T therapy. Seven (8.43%) patients developed isolated CNS relapse after CAR-T cell therapy, all of which were heavily pre-treated with multiple lines of chemotherapy prior to CAR-T cell therapy, and one of them had received autologous stem cell transplant before infusion of CAR-T cells. Only 4 of 7 patients with isolated CNS relapse achieved CR (n=4), and the median time to isolated CNS relapse from CAR-T cell infusion was 6.3(range, 1.53 to 25.8) months. Thirty-seven patients (46.2%) developed systemic recurrence without CNS involvement, with the median time from CAR-T to relapse of 3.2 (range, 0.467-30.8) months. The other 36 patients remained in remission at the last follow-up. During the median follow-up of 13.5 months of the whole cohort, the number of recorded deaths in isolated CNS relapse, systemic only recurrence, and no relapse patients was 5 (71.4%), 22 (59.4%), 1(2.78%), respectively. The 1-year OS and 1-year PFS of patients with isolated CNS recurrence was significantly lower compared to the no relapse group (OS, 35.7% [95%CI 12.1-100] vs 97.22% [95%CI 92-100], P&amp;lt;0.001; PFS, 28.6% [95%CI 8.86-92.2] vs 100% [95%CI 100-100], P&amp;lt;0.001), but showed no difference compared to the systemic only recurrence group(OS, 53.3% [95%CI 39.3-72.4], P=0.79; PFS, 13.0% [95%CI 5.53-30.5], P=0.17). Conclusion In conclusion, patients with CNS relapse showed significant difference in 1-year outcomes with no relapse group but not systemic relapse group in our study, which suggested the importance of effective prophylaxis against CNS relapse. Further investigation is necessary to better define the characteristics of patients who will benefit from prophylaxis. Acknowledgements We gratefully thank all the participation from patients enrolled in clinical trials (ChiCTR1800015575; NCT03118180; NCT04532281; NCT04532268; NCT04213469).

Day +3/Day 0 Ferritin Ratio: A Simple Point-of-Care Index Predictive of Severe Immune Effector Cell-Associated Neurotoxicity Syndrome after CD19 CAR T-Cell Therapy

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is transforming the management of patients with relapsed/refractory (R/R) B- and plasma-cell malignancies. However, a major limitation of CAR T-cell therapy is the development of severe, sometimes life-threatening, toxicities such as immune effector cell-associated neurotoxicity (ICANS) and cytokine release syndrome (CRS). While the management of CRS has dramatically improved with the early use of tocilizumab and corticosteroids, how to best predict and treat ICANS remains poorly understood, with up to 30% of patients developing severe ICANS after CD19 CAR T-cell therapy. Since we had previously observed features overlapping with hemophagocytosis lymphohistiocytosis in patients with severe CRS and ICANS, we hypothesized that the early kinetics of ferritin and soluble IL-2 receptor alpha [sIL-2Rα] post infusion could predict the development of severe ICANS. Methods: We retrospectively analyzed data from 195 patients with R/R B-cell malignancies previously treated on a phase I/II clinical trial (NCT01865617) of an investigational defined 1:1 CD8+:CD4+ composition CD19 CAR-T product (JCAR014; training set). CRS and ICANS severity were graded using Lee 2014 and CTCAE 4.03 criteria, respectively. Logistic regression was used to predict the occurrence of severe ICANS. Ferritin was measured using an enzyme immunoassay. sIL-2Rα was measured using Luminex®. Goodness of fit was assessed using the Akaike information criterion (AIC). Independent test set included 203 patients treated at our center with the standard-of-care CD19 CAR T-cell products axicabtagene or lisocabatagene maraleucel. Discrimination and calibration were assessed in both the training and test set. Results: In the training set (n=195), median age was 55 (range: 20-76). Disease type was as follows: acute lymphoblastic leukemia, n=64 (32.8%); B-cell non-Hodgkin lymphoma, n=82 (42.1%); chronic lymphocytic leukemia, n= 47 (24.1%). We observed any grade and grade ≥3 CRS in 134 (68.7%) and 27 (13.8%), respectively, and any grade and grade ≥3 ICANS in 72 (38.8%) and 37 (19.0%), respectively. Longitudinal analyses of serum ferritin (Figure 1) and sIL-2Rα values using locally estimated scatterplot smoothing suggested that the rate of increase of both analytes was markedly higher in patients with grade ≥3 ICANS compared to grade 0-2 ICANS. We applied univariate logistic regression to estimate the association between serum ferritin levels at early timepoints, select ratios of ferritin and sIL-2Rα values, and the occurrence of severe ICANS. The strongest association was observed between the ferritin day +3/day 0 ratio and severe ICANS (OR per log10 increase, 175; 95% CI, 19.1-2668; p &amp;lt; 0.001). We could further improve our model by modeling the ferritin day +3/day 0 ratio with a restricted cubic spline to allow for non-linear effects (unsplined model, AIC = 129, C-index, 0.74; splined model, AIC = 127, C-index, 0.78). Ferritin day +3/day 0 ratios of 3, 5, and 10 were associated with an 81%, 95%, and 99% probability of grade ≥3 ICANS, respectively (Figure 2). The splined model was associated with near-perfect calibration corrected for optimism using bootstrapping. We next evaluated the predictive ability of the ferritin day +3/day 0 ratio to predict severe ICANS in our independent test set. Predictions in the test set based on our splined model retained good discriminative ability with a C-index of 0.64, although with suboptimal calibration. In a refitted logistic regression model in the test set, the ferritin day +3/day 0 ratio remained strongly associated with the development of severe ICANS (OR = 18.8 per log10 increase, 95%CI, 2.48-142.01, p = 0.004). Conclusion: We validated the ability of a simple point-of-care index (day +3/day 0 ferritin ratio) to predict the development of severe ICANS after CD19 CAR T-cell therapy in our training and test set. Since serum ferritin levels can be quickly measured by most clinical laboratories, the day +3/day 0 ferritin ratio could become a practical and useful tool for practitioners taking care of CAR T-cell patients. The early identification of patients at high risk of developing severe ICANS days prior to symptoms onset could enable the evaluation of targeted prophylactic interventions in future clinical trials. We plan to refine our modeling approach to further improve the calibration in test set.

Prophylactic or Preemptive Donor-Derived CD19 CAR-T Cell Infusion for Preventing Relapse in High-Risk B-ALL after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Relapse remains a significant challenge in the management of high-risk B-cell acute lymphoblastic leukemia (B-ALL) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Existing post-transplant relapse prevention strategies for high-risk B-ALL patients after transplantation, particularly in cases of Philadelphia chromosome-negative B-ALL, have limitations and suboptimal efficacy. This study aims to evaluate the efficacy and safety profile of prophylactic or preemptive infusion of donor-derived CD19 chimeric antigen receptor T (CAR-T) cells in high-risk B-ALL patients after allo-HSCT. Methods: Between May 2017 and July 2022, high-risk B-ALL patients with minimal residual disease (MRD) positive or MRD negative status after allo-HSCT were enrolled in clinical trials of donor-derived CD19 CAR-T cell therapy (ChiCTR 2000041025 and ChiCTR-ONN-16009862). The primary outcome was the cumulative incidence of relapse (CIR). Secondary outcomes were progression-free survival (PFS), overall survival (OS) and CAR-T cell treatment-related toxicities. Results: A total of 25 high-risk B-ALL patients who received infusion of donor-derived CD19 CAR-T cells after allo-HSCT were enrolled in the CAR-T cell group, including 8 patients with MRD and 17 patients without MRD. A contemporaneous cohort of 43 high-risk B-ALL patients who did not receive CAR-T cell therapy in MRD-positive or MRD-negative status after transplantation was also included as a control group. The median age of patients enrolled in the CAR-T group was 29 years (range: 9-60 years). The median infused CAR-T cell dose was 2.5×10^6/kg (range: 1.0-5.0×10^6/kg), with a median time from transplantation to CAR-T cell treatment of 122 days (range: 45-315 days). The median follow-up duration was 23.6 months (range: 8.3-60.6 months). Of the 25 patients in CAR-T group, eight MRD-positive patients achieved MRD-negative remission after CAR-T cells treatment (Figure1A). The two-year cumulative incidence of relapse in the CAR-T cell group was significantly lower at 4.8% (95% CI: 0.0%-12.6%) compared to the control group's incidence of 26.6% (95% CI: 11.4%-41.8%, P = 0.03，Figure1B). Additionally, the CAR-T cell group exhibited a higher two-year PFS rate [86.3% (95% CI: 71.8%-100%)] compared to the control group [63.5% (95% CI: 48.0%-79.0%), P = 0.04]. However, the two-year OS rates were comparable between the CAR-T cell group [91.0% (95% CI: 79.0%-100%)] and the control group [79.1% (95% CI: 66.0%-92.2%), P = 0.20]. Adverse events were well tolerated, and no patients experienced grade 3-4 cytokine release syndrome or any grade of immune effector cell-associated neurotoxicity syndrome. Hematological cytopenia was the most severe CAR-T cell related toxicity, but the toxicity was manageable, and only three patients experienced acute graft-versus-host disease following CAR-T cell infusion. Conclusions: Prophylactic or preemptive infusion of CAR-T cells after allo-HSCT demonstrated a reduced incidence of relapse and acceptable adverse events in high-risk B-ALL patients. These findings highlight the potential of CAR-T cell therapy as a promising strategy for relapse prevention in high-risk B-ALL patients after allo-HSCT.

Clonal Hematopoiesis Is Associated with Severe Cytokine Release Syndrome in Patients Treated with Chimeric Antigen Receptor T-Cell (CAR-T) Therapy

Background: Among patients receiving CD19 or B-cell maturation antigen (BCMA) CAR-T therapy, inflammation pre- and post-CAR-T infusion is attributed in the development of toxicities such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and prolonged cytopenias. Clonal hematopoiesis (CH), the clonal expansion of hematopoietic stem cells with somatic mutations, has been associated with inflammatory disorders such as rheumatoid arthritis and graft-versus-host disease, as well as clonal cytopenias of undetermined significance. In the setting of CAR-T, others have reported an association between CH and both ICANS and treatment-associated myeloid neoplasms. Herein, we examined the prevalence of pre-CAR-T CH in a predominantly transplant-naïve cohort of recipients with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM), and assessed the relationship between CH and CAR-T related outcomes including CRS, ICANS, prolonged cytopenia, progression-free survival (PFS), and overall survival (OS). Methods: This study included 62 patients with NHL or MM who underwent CD19 or BCMA CAR-T therapy from 2017 to 2022 at City of Hope and had available pre-CAR-T cryopreserved peripheral blood mononuclear cells (PBMCs). DNA was isolated with QIAamp DNA Mini Kit (Qiagen) from PBMC samples (94% collected &amp;lt;30d of CAR-T infusion), on which we performed targeted exome sequencing (108 pre-defined gene panel with 1000x sequencing depth) to determine the presence of CH (variant allele frequency [VAF] ≥2%). Multivariable logistic regression was used to examine the association between CH (Yes/No) and absolute neutrophil count (ANC) recovery at day +60 (Y/N), maximum grade CRS and ICANS, grade &amp;lt;2 vs 2+, and OS and PFS at 1y. Covariables considered were age at CAR-T, baseline ANC, sex, race, CAR-HEMATOTOX (Rejeski et al. 2021), bridging therapy (Y/N), and number of prior lines of therapy. ANC recovery was defined as grade 2 or better for 3 measurements without growth factor support &amp;gt;2 weeks. Those without ANC recovery and who started new treatment before day +60 were excluded from that analysis. Results: Overall, the median age was 64y (range 21y-85y); 37 (60%) had de novo diffuse large B-cell lymphoma, 17 (27%) had transformed follicular lymphoma, 2 (3%) had MM, 2 (3%) had mantle cell lymphoma, and the remaining patients had other transformed indolent lymphomas. Median prior lines of therapy was 3 (1-12), and only 6% had previously undergone autologous hematopoietic cell transplantation. Systemic bridging therapy prior to CAR-T was required in 27 (44%), and 8 (13%) received radiation as bridging. All received fludarabine and cyclophosphamide for lymphodepleting chemotherapy. Axicabtagene ciloleucel was the most common (76%) CAR-T product, followed by tisagenlecleucel in 16% of patients. Overall, 51 (82%) and 29 (47%) patients experienced CRS and ICANS, respectively. had delayed ANC recovery. One-year PFS was 50% and 1y OS was 73%. Clonal hematopoiesis: 15 (24%) patients had at least one pathogenic CH mutation; 4 (6.5%) had ≥2 CH mutations. Of the 28 CH mutations identified, the most common were DNMT3A (18%), CHEK2 (11%), PMS2 (7%) and ASXL1 (7%); 29% of mutations had VAFs &amp;gt;10%. Outcomes after CAR-T: Patients with CH were significantly more likely to develop grade ≥2 CRS (60% vs. 28%, p=0.03) compared to those without CH (odds ratio [OR] 3.9, 95% CI 1.2-13.2; p=0.027). Accounting for baseline ANC (which was unexpectedly higher among the CH cohort and associated with delayed ANC recovery, p=0.02) patients with CH did not have a significantly different rate of delayed ANC recovery compared to those without CH (adjusted OR 0.37, 95% CI 0.09-1.5; p=0.17). There was no association between CH and ICANS, nor with 1y PFS and OS. Conclusion: CH was frequent (24%) in this cohort of CAR-T recipients and was associated with a higher risk of ≥2 CRS after CAR-T. Additional validation studies are currently underway, which may set the stage for consideration of pre-CAR-T CH as a biomarker for risk stratification towards more proactive CRS prophylaxis Acknowledgements: Funding through the CoH Hematological Malignancies Program Pilot Project Award. The authors acknowledge the work provided by CoH Center for Informatics, notably Research Informatics, and the utilization of the POSEIDON platform and the Honest Broker process.

Impact of Treatment with Inotuzumab Ozogamicin before or after Chimeric Antigen Receptor T-Cell Therapy in Children with Relapsed/Refractory Acute Lymphoblastic Leukemia

Background: Inotuzumab (InO), a monoclonal antibody targeting CD22 conjugated to calicheamicin, is widely used for relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) with good salvage rates (O'Brien, JCO 2022, Bhojwani, Leukemia 2019). Feasibility data on its use as a bridge to Chimeric Antigen Receptor T-cell therapy (CART) or as salvage therapy post CART is limited. Concern has been raised that InO given prior to leukapheresis might impact the quality of T-cell collection and, when used for bridging therapy prior to CART, could result in insufficient B-cell antigen load at the time of CART infusion, therefore reducing efficacy. Methods: Data of patients who received InO monotherapy on Children's Oncology Group study AALL1621 Cohort 1 and who received any form of CART prior to study enrollment or within 1 year of coming off protocol therapy, irrespective of other anti-leukemic treatments, were collected retrospectively using a standardized Case Report Form. Results: Forty-eight patients received InO on AALL1621; 3-year overall survival (OS) rate was 35.4±6.9%. Thirteen received InO prior to CART and thirteen received InO post CART including 1 patient who received CD19 CART prior to InO and CD22 CART post InO (Table 1). InO prior to CART (11 CD19 CART, 1 CD22 CART, 1 unknown): Seven patients went directly to CART post InO, 5 received intervening chemotherapy, and 1 had an intervening transplant with relapse post. Nine patients received tisagenlecleucel and 3 received an investigational CART. Patients were infused CART 1-150 days (median 45 days) after coming off protocol therapy. At CART infusion, 10 patients had detectable ALL, only 2 had no CD19 expressing cells at infusion (ALL or normal B-cells) and 1 unknown. At post CART bone marrow evaluation on day 28, 7/12 (58.3%) were in a minimal residual disease (MRD) negative (&amp;lt;0.01%) remission, 1 had MRD=0.021%, 1 had unknown response. Three had residual disease including the single patient who received CD22-targeted CART. Four MRD negative patients who received CART post-InO are alive without relapse at a median of 3.8 years post CART and 1 is alive with a subsequent relapse. At 3 years post CART infusion OS is 53.9±13.8%. T-cells were collected immediately following 3-9 doses of InO in 8/13 (61.5%) patients (1-6 months from date of enrollment on the study), 2 were collected prior to InO and three had unknown collection dates. All had a CART product manufactured successfully. Post CART 7/13 patients did not develop thrombocytopenia (&amp;lt; 50,000/uL); 5 who developed thrombocytopenia had measurable ALL at day 28 post CART or soon after. One other patient went to planned transplant post CART and remained with low platelets and ANC. Almost all patients (11/13) had ANC &amp;lt; 500/uL post CART, of whom 3 did not recover including two with persistent ALL. InO Post CART (11 CD19-targeted CART, 2 CD22-targeted CART): Of the 13 patients who received InO post CART therapy, 7/13 (53.8%) achieved an MRD-negative complete remission (CR) to CART but then developed detectable disease 2-36 months post CART. Five did not have a CR post-CART, 4 of whom enrolled on AALL1621 within one month; 1 received intervening chemotherapy. One had an unknown response. On univariate analysis CR rate with InO therapy was no different for those who received CART prior to InO than those who did not. As well, risk of a dose limiting toxicity event (DLT) or developing sinusoidal obstructive syndrome (SOS) during InO therapy or during subsequent transplant after InO was not different in patients with CART exposure prior to AALL1621 enrollment. Conclusion: The ability to collect and manufacture CART cells was feasible within weeks of receiving InO. Response to CART post InO was lower than retrospective cohorts (Ceolin, Leukemia 2022) but 3-year OS post CART was consistent with published CD19-targeted CART trials at 53.9% (Maude, NEJM 2018; Pasquini, Blood Adv 2020). InO did not appear to impact durability of remission in those who attained an MRD negative remission with CART. Given most patients had a CD19 target due to disease presence at the time of CART infusion, differential response to CART based upon antigen could not be analyzed. Prior InO exposure did not appear to worsen cytopenias post CART when compared to published CART trials and was associated with a comparable 3-year OS. As well, CART exposure prior to InO did not appear to effect response or increase the risk of DLT or SOS during subsequent InO therapy.

CAR T-Cell Therapy Combined with PD-1 Inhibitors Significantly Improve the Efficacy and Prognosis of r/r DLBCL with TP53 Alterations

More than 40% of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) eventually develop relapsed or refractory (r/r) disease and typically have poor outcomes. Autologous chimeric antigen receptor T-cell therapy (CART) has emerged as a crucial second line treatment option for r/r DLBCL, but 60-70% of patients still relapse or are refractory. Recent research has shed light on the role of TP53 alterations (mutation or deletion) in r/r DLBCL patients who do not respond well to CART cell therapy. The findings in this study will show that the combination of CART with PD-1 inhibitors (ICIs) had a significant impact on the efficacy and prognosis of r/r DLBCL patients with TP53 alterations. This retrospective analysis included adult DLBCL patients who were treated at Shanghai Tongji Hospital of Tongji University with autologous CD19 (N=20) or CD20 (N=2, patient 6 and 7, Fig.1 A) CART-cell therapy between January 1, 2019, and September 30, 2022. All enrolled patients provided their informed consent to participate in the study. The protocol of the study was reviewed and approved by the Institutional Review Board of the National Cancer Institute, ensuring ethical practices were followed. A total of 22 patients with TP53 alterations undergoing CD19-CART therapy were included, of which 2 patients received CD20 CART cell after recurrence of CD19 CART cell therapy. The total dose of CD19 or CD20-CAR T-cell is between 1- 3x10^6/kg. Sintilimab/Tislelizumab was used as PD-1 inhibitors (3 mg/kg in patients weighing &amp;lt;60 kg or 200 mg in patients weighing ≥60 kg) every three weeks within 6 weeks after CAR-T infusion. 1 case had grade 3-4 Cytokine Release Syndrome (CRS) and no case had CART-cell-related encephalopathy syndrome (CRES). In Table 1, the patients are divided into two groups. One group received CART cells combined with PD-1 inhibitors (ICI+), while the other group received CART cells only (ICI-). The median ICIs treatment time after CART infusion was 17 days (IQR 11-38 days). Baseline factors such as age, gender, disease severity, previous treatment history and etc. had no substantial differences between the groups. Treatment regimen and the subsequent response were shown in Fig.1 A and B. Patient 1-9 received ICIs treatment after CART cell therapy, of which 8 cases (88.9%) showed the best objective response rate (ORR) at day 90 after CART infusion (Fig.1B) and 6 cases (66.7%) achieved complete remission (CR). In comparison, among the 13 individuals who were treated with CART alone, only 3 cases (23.1%) showed the best ORR at day 90 after CART infusion and only 1 case (7.7%) achieved complete remission. We found a significant difference between the two treatment groups by performing a Chi-square test (P=0.009). Our results suggest that the addition of PD-1 inhibitors to the CART regimen significantly improves the treatment outcome in patients with TP53 alterations. In Fig.1 C and D, we show that the median progression-free survival (PFS) was NR (not reached) vs. 1.7 months (95% CI:1.0 month-2.3 months) and the median overall survival (OS) was NR vs.10.9 months (95% CI: 3.8 months-18.1 months) in the ICI+ and the ICI- groups. The p-values for PFS and OS was 0.001 (HR=0.18, 95% CI: 0.07-0.47) and 0.003 (HR=0.15, 95% CI: 0.05-0.42) between the two groups. These results suggest that patients in the combination treatment group experienced less disease progression and were more likely to survive longer. Overall, our study demonstrates the potential benefits of combining immunotherapies, specifically CART with PD-1 inhibitors, in the treatment of relapsed or refractory DLBCL patients with TP53 alterations.

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.

Updated Results of the Phase I BALLI-01 Trial of UCART22 Process 2 (P2), an Anti-CD22 Allogeneic CAR-T Cell Product Manufactured By Cellectis Biologics, in Patients with Relapsed or Refractory (R/R) CD22+ B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Introduction: UCART22 is a genetically modified allogeneic T-cell product manufactured from healthy donor cells. Donor-derived T-cells are transduced using a lentiviral vector to express the anti-CD22 chimeric antigen receptor (CAR) and are further modified using Cellectis' TALEN ® technology to disrupt the T-cell receptor alpha constant ( TRAC) and CD52 genes to minimize risk of graft-vs-host disease (GvHD) and allow use of an anti-CD52 antibody for lymphodepletion (LD). Preliminary results from patients treated with UCART22 manufactured by a CMO (Process 1 (P1)) showed that UCART22 was well-tolerated and meaningful responses were achieved at the highest dose level (DL 3; 5 x 10 6 cells/kg). The fludarabine, cyclophosphamide, and alemtuzumab (FCA) LD regimen was also shown to extend host lymphocyte suppression and improve UCART22 expansion versus fludarabine and cyclophosphamide (FC) alone (Boissel N, et al. EHA 2023). We now report updated results from the BALLI-01 study that includes the first patients treated with UCART22 Process 2 (P2) manufactured by Cellectis Biologics. Methods: The primary endpoints are safety, tolerability, and determining the MTD/RP2D of UCART22. Additional endpoints are anti-leukemic activity and expansion of UCART22. Eligibility criteria include age 15‒70y, B-ALL blast CD22 expression ≥ 70%, and ≥ 2 prior treatment regimens. After FCA (F 30 mg/m 2 × 3d, C 0.5g/m 2 × 3d, A 20 mg/d × 3d) LD regimen, pts received a single infusion of UCART22-P2. In vitro comparability assays suggested that UCART22 P2 was more potent than UCART22 P1, so dose escalation with UCART22 P2 started at DL2 (1 x 10 6 cells/kg) compared to the highest studied dose DL3 (5 x 10 6 cells/kg) with UCART22 P1. Results: As of 01 July 2023, 3 pts were enrolled into the first UCART22 P2 cohort at DL2. Pt 1 is a 17yo female with B-ALL with a hypodiploid karyotype and a germline TP53 mutation whose disease had previously failed to respond to multiagent chemotherapy, blinatumomab (blina), inotuzumab (ino), venetoclax (ven), allogeneic hematopoietic stem cell transplantation (HSCT), and autologous CD19 CAR T-cell therapy (CAR19) x2. Pt 2 is a 68yo female with Ph-negative B-ALL who relapsed with CD19-low disease after multiagent chemotherapy, ino, and blina. Pt 3 is a 27yo male with B-ALL with an ABL2 fusion who had failed multiagent chemotherapy, ino, blina, TKIs, and an experimental autologous CAR19. UCART22 P2 administered after the FCA LD regimen was well tolerated. No DLTs or ICANS were observed. Cytokine release syndrome (CRS) occurred in 2/3 (67%) pts, with one G1 that resolved without treatment and one G2 that resolved after tocilizumab x1. There was one G5 sepsis SAE at D40 considered related to UCART22 P2 and FCA LD in Pt 1. Responses were assessed beginning on D28. Up to FC/FCA-DL3 with 18 pts treated with UCART22 P1, 1 CR, 4 CRi, and 2 morphologic leukemia-free states (MLFS) were observed, with 3 responses occurring out of 6 (50%) patients treated with FCA-DL3 (previously reported, EHA 2023). For UCART22 P2, FCA-DL2, 2/3 pts (67%) responded: Pt 2 achieved an MRD neg CR lasting over 84 days after UCART22 infusion; Pt 1 achieved an MRD negative MLFS up to D40. Pt 3 was refractory to treatment, however this pt received bridging therapy with dasatinib for his ABL2 fusion, and on Day -1, only 47% of the leukemic cells expressed CD22 (down from 88% at screening). UCART22 P2 expansion was observed by flow cytometry in peripheral blood with peak of 79 cells/μl in Pt 1 and 225 cells/μl in Pt 2, both at D11, with predominantly CD8 cells expanding. Inflammatory markers such as ferritin, IFN-γ, TNF-α and IL-6 levels increased more than 3-fold, correlating with UCART22 P2 expansion and CRS. Summary : In vitro comparability studies indicated that UCART22 P2 was more potent than UCART22 P1, and this was suggested clinically, as there was a 67% response rate at DL2 with UCART22 P2 compared to 50% at DL3 with UCART22 P1. Use of UCART22 P2 did not lead to any grade ≥3 CRS, and no DLTs or ICANS was observed. UCART22 P2 expansion was seen in the two responders, which closely correlated with CRS and changes in inflammatory markers. These data support the safety and preliminary efficacy of UCART22 P2 in this poor-risk R/R B-ALL population. The study continues to enroll pts treated with UCART22 P2 at dose level 2i (2.5 x 10 6 cells/kg), and updated data will be presented.

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.