T-cell Production Research Articles

Phase I Study of a CD19-Directed CAR-T Cell Therapy for Relapsed/Refractory Mantle Cell Lymphomas(MCL)

Background: IM19 is an autologous, CD19-directed, chimeric antigen receptor (CAR) T-cell product, which is manufactured in China using a commercial process. We aimed to evaluate the efficacy and safety of IM19 in patients with relapsed or refractory mantle cell lymphomas in a Phase I clinical study. Methods: A phase 1 clinical trial has been launched to evaluate the safety and efficacy of IM19 for the treatment of relapsed/refractory MCL. We enrolled adult patients (aged ≥18 years) with relapsed or refractory MCL who had received at least 2 prior treatment regimens(including BTK inhibitor). Patients were assigned to one of two target dose levels of IM19 as they were sequentially tested in the trial (100×10⁶ CAR+ T cells [one or two doses], 200×10⁶ CAR+ T cells [one or two doses]). Leukapheresed patients could receive bridging chemotherapy based on investigator assessment of disease burden and tumor progression risk. Lymphodepleting (LD) chemotherapy was administered over 3 days, including fludarabine (30 mg/m2 i.v. daily) and cyclophosphamide (300 mg/m 2 i.v. daily), followed 2 days later by intravenous infusion of IM19 at the assigned dose. The first three subjects in each dose group will receive the second LD chemotherapy and infusion of IM19 CAR-T cells for consolidation treatment (with the same dosage as the first treatment) if the efficacy evaluation after the first infusion shows no progression and no DLT occurs. The investigators can determine the start time of consolidation treatment based on the patient's tolerance, but no later than 60 days after the first infusion. Efficacy was assessed at 1, 3, 9, 12, 18, and 24 months post first infusion. Safety events were monitored from beginning of LD through 2 years follow-up. The primary endpoints were adverse events, dose-limiting toxicities, and the objective response rate. Results: Between Mar 3, 2022, and May 5, 2023, Five patients underwent leukapheresis for manufacture of CAR+ T cells (IM19) and all received bridging chemotherapy during the production of IM19. ALL 5 patients received LD and first infusion of IM19 at a dose of 100*10^6 CAR+ cells and no DLTs were observed. CRS was reported only in 1 patient(grade 1) and no patients developed ICANS. ALL 5 patients achieved CR on 28 days after first infusion. The median maximum CAR-T cells expansion (Cmax) was 156×10⁶ CAR+ cells/L, and median area under the curve from 0-28 days post-first infusion (AUC0-28d) was 860 day×CAR+ cells/L. 2 patients received the second LD chemotherapy and infusion of IM19 CAR-T cells for consolidation treatment (with the same dosage as the first treatment) at 44th days post first infusion. No DLTs，CRS or ICANS were observed after second infusion. Meanwhile, CAR-T cells can not be detected at 1, 4, 7, 10, 14, 21 and 28 days post second infusion by Flow cytometry in 2 patients. One patient maintained complete remission after 9 months of follow-up and another patient experience PD at 7th month after first infusion. For three patients who received single infusion of IM19, one maintained complete remission after 6 months of follow-up，one progressed at 3th month after infusion and one are still waiting for second efficacy evaluation. Conclusions: Initial data from this Phase I study demonstrate that use of IM19 resulted in a high objective response rate, with a low incidence of grade 3 or worse cytokine release syndrome and neurological events in patients with relapsed or refractory MCL. We observed that CAR-T cells cannot expand in vivo after secondary infusion. Therefore, secondary infusion may not be necessary.

CD4+/CD8+ Selection of Anti-AML ENG-T Cells Affects Long-Term T-Cell Persistence in Vitro and Survival in an AML Xenograft Model

Background: Immunotherapies have shown dramatic anti-cancer efficacy in pediatric hematopoietic malignancies. Despite initial disease clearance, relapse rates are high, demanding product improvement. Manufacture of engineered cellular immunotherapies involves expansion of effector cells from patient or donor peripheral blood mononuclear cells (PBMCs) ex vivo prior to patient infusion. Expansion methods in pre-clinical studies of engineered T-cell therapies often call for activating T cells from whole PBMCs using anti-CD3 and anti-CD28 co-stimulation. However, clinical production of T-cell therapies has shifted towards selecting CD4+ and CD8+ cells prior to activation due to higher product efficacy seen in clinical trial. Many acute myeloid leukemias (AML) are CD4+, introducing the risk of blast contamination during CD4+ selection. We questioned whether CD8+ T cells alone could be used to manufacture our clinical T cell product and sought to determine how selection affected our T cells. Understanding the biological impact of manufacturing changes between pre-clinical and clinical production is imperative for clinical translation. Methods: We have engineered T cells to secrete a CD123xCD3 binding bispecific engager molecule (CD123-ENG) to treat AML. Primary T cells were isolated from PBMCs, selected with microbeads, activated, and transduced with replication incompetent retroviral vectors. Unselected T cells were compared to CD4+, CD8+, or CD4+ and CD8+ selected T cells. T-cell transduction was confirmed with flow cytometry. Expression and secretion of CD123-ENG was confirmed with ELISA. Antigen-specific T-cell activation was determined by quantifying cytokine secretion in the presence of CD123+ target cell lines. Short-term cytotoxicity was examined in vitro against CD123+ target cell lines in 18-hour bioluminescence assays. T-cell serial cytotoxicity and persistence were tracked in 10-day serial stimulation studies using an IncuCyte live cell imaging system. Cytokines present during T-cell manufacture and serial stimulation were detected and quantified with Luminex multiplex assays. Immunophenotype and exhaustion markers were analyzed using flow cytometry. In vivo anti-AML cytotoxicity and T-cell persistence were determined in an AML xenograft model. Results: In our studies, unselected, CD4+, CD8+, and CD4+/CD8+ selected CD123-ENG T cells showed no differences in activation, cytotoxicity, or immunophenotype when evaluated in short-term assays. Mice harboring human AML treated with CD4+/CD8+ selected CD123-ENG T cells had improved survival compared to mice treated with unselected and CD8+ selected CD123-ENG T cells. CD4+ T cells alone provided the best survival advantage. In addition, CD4+/CD8+ selected T cells had longer persistence when challenged by serial target cell stimulations in vitro. T cells expanded from whole PBMCs were exposed to cytokines likely to emanate from non-T cells during early manufacture. These cytokines were characteristic of those produced by myeloid cells in an inflammatory environment, and may explain functional differences observed in vivo. Conclusions: Durable anti-cancer response in patients is associated with T-cell persistence and CD4+/CD8+ pre-selection of CD123-ENG may therefore improve remission duration in patients. CD4+ and CD4+/CD8+ selected T cells provided the longest survival in vivo, dissuading us from further investigating CD8+ CD123-ENG as an AML treatment. We are actively exploring additional mechanistic determinants of the observed phenotypic differences with and without selection. Optimizing cellular immunotherapy will improve patient outcomes. It is essential that engineered cellular research products intended for clinical translation be produced by the same methods used for clinical manufacture to increase the predictive power of pre-clinical data. The use of biological correlatives to predict T-cell product function will better support successful translation. Acknowledgements: This research was funded in part by NCI Contract No. HHSN26120150003I/HHSN26100077.

Underrepresentation of Older Adults in Clinical Trials of Chimeric Antigen Receptor (CAR) T-Cell Therapy for Hematologic Malignancies: An FDA Analysis

Background: As the population ages, the prevalence of hematologic malignancies in older adults will continue to increase. Older adults, especially older than 75 years, are routinely underrepresented in clinical trials evaluating hematologic malignancies, largely due to access and logistics, comorbidities, toxicity concerns, and eligibility restrictions. Given the recent approvals of multiple CAR T-cell therapies for patients with hematologic malignancies, we evaluated the rates of enrollment of older adults (age ≥ 65 years) in registrational trials of CAR T-cell therapies submitted to the U.S. Food and Drug Administration (FDA). Methods: We conducted a pooled analysis of the registrational trials submitted to the FDA between 2017 and 2021 that supported approvals of autologous CAR T-cell therapies in patients with hematologic malignancies. Datasets submitted with the biologic license applications were pooled and analyzed descriptively for demographics, disease characteristics, and efficacy outcomes. Patients between the ages of 65 and 74 and age 75 and above were analyzed separately. Results: Thirteen registrational trials evaluating six CAR T-cell products in 1,319 patients with hematologic malignancies were submitted to FDA. The median age at enrollment was 60 years (range: 3 to 86). In total, 7% (88) of patients were 75 years and older, 28% (366) were between the ages of 65 and 74 years, and 66% (865) were under the age of 65 years. The hematologic malignancies included large B-cell lymphoma (59%), multiple myeloma (15%), follicular lymphoma (13%), B-cell acute lymphoblastic leukemia (9%), and mantle cell lymphoma (5%). Response rates by age group and histology are shown in Table 1 and Table 2. Conclusions: Older adults, particularly those aged 75 years or older, were underrepresented in the trials supporting FDA approval of CAR T-cell therapies considering the predominant hematologic malignancies included in this pooled analysis and the median age of diagnosis of these diseases in the U.S. population. Although clinically meaningful efficacy was achieved across age groups and diseases, more data are needed to determine the impact of age on efficacy outcomes in specific malignancies. In this analysis, data were limited among patients 75 years and older in all histologies except large B cell lymphoma. Work to evaluate the impact of age on safety and tolerability outcomes is ongoing. To improve accrual, greater efforts to understand and reduce barriers to clinical trial enrollment for older adults are needed, especially given the expanding use of CAR T-cell therapies and their toxicity profiles.

Improving Anti-Tumor Efficacy of CAR T-Cell Therapy for Acute Myeloid Leukemia (AML): Results from a Multi-Drug Interaction Screening Approach

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled proliferation and impaired differentiation of myeloid progenitor cells. With a 5-year overall survival of approx. 30%, the prognosis of patients with acute myeloid leukemia remains poor, in particular for those with relapse or refractory disease. The adoptive transfer of T-cells genetically modified to express a chimeric antigen receptor (CAR) have shown impressive remission rates in patients with B-cell lymphoid malignancies. In contrast, CAR T-cell therapy for AML has induced durable remission only in a minority of patients so far, highlighting the need for strategies to improve anti-tumor efficacy and persistence of AML-specific CAR T-cell products. Adapting an established high throughput screening platform, we aimed to identify compounds with the potential to enhance CAR T-cell mediated cytotoxicity through interaction with tumor and/or CAR T-cells. Methods: We established a high throughput platform to determine the cytotoxicity of four different AML-specific CAR T-cell products targeting CD33 (CD33.CD28.41BB.zeta), CD70 (CD27.zeta), CLL1 (CLL1.CD28.zeta) and EMR2 (also known as ADGRE2; EMR2.41BB.zeta) in the presence of 32 compounds at five different concentrations in a 10,000-fold concentration range. CAR T-cells were generated by transduction of activated T-cells derived from peripheral blood mononuclear cells (PBMCs) of three healthy donors with a gamma retroviral vector containing the different CAR constructs. On day 11 after activation, CAR T-cells and non-CAR-transduced control T-cells were incubated with luciferase-expressing target cells at an effector-target ratio of 1:4 in 384-well plates. Viability of target cells cultured with CAR T-cells, control T-cells or in the absence of T-cells was calculated after 24 and 48 hours using a luciferase signal intensity assay. Combinatorial toxicities were calculated with the Bliss Independence model for synergistic effect and the highest single agent model for additive combination effect. Results: In quality analysis, the intensity of luminescence signals in control wells (tumor only, non-CAR-transduced control T-cells) of different plates was similar and principal component analysis (PCA) of samples derived from different days (batches) did not show significant differences ruling out relevant edge or batch effects that could negatively impact the screening results. Of all tested compounds birinapant, a peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) and inhibitor of apoptosis protein (IAP) family proteins and the hypomethylating agent (HMA) decitabine showed the strongest improvement of CAR T cell cytotoxicity in an additive but also in a synergistic manner. These effects were evident in CAR T-cell products derived from all different T cell donors without significant differences. Depending on the structural composition of the CAR construct, the strongest drug-induced improvement of CAR T-cell cytotoxicity was seen either after 24 or 48 hours. The third generation CAR T- cell construct (CD33.CD28.41BB.zeta) showed enhanced cytotoxicity already after 24 hours, whereas significant anti-leukemic efficacy of CD27.zeta, CLL1.CD28.zeta and EMR2.41BB.zeta CAR T-cells became evident at the later 48 hours timepoint. Importantly, co-treatment with birinapant and decitabine particularly enhanced the cytotoxicity of EMR2-specific CAR T-cells which exhibited the lowest baseline anti-tumor efficacy compared to all other CAR constructs highlighting the potential of distinct chemical compounds to improve the functionality of AML-specific CAR T-cells. Conclusion: Our data suggest that high throughput drug interaction screens are a reliable approach to investigate the impact of chemical compounds on the functionality of AML-specific CAR T-cell products. HMA and SMAC mimetics are promising candidates that may potentially enhance the cytotoxicity of CAR T-cells. Ongoing studies are evaluating the effect of HMAs and SMAC mimetics on CAR T-cell proliferation and anti-tumor efficacy against AML cell lines and primary AML patient samples upon serial antigen stimulation. Furthermore, in-depth proteogenomic characterization to identify molecular signatures of enhanced functionality are currently being performed.

High Body Mass Index Is Associated with Favorable Outcome in Younger Patients Receiving CD19 CAR T-Cell Therapy for B-Cell Lymphoma: A Retrospective Study from the EBMT Transplant Complications Working Party

Introduction: Obesity is a major health care burden and has been linked to numerous adverse outcomes including alterations of antitumor immunologic responses. Patients with high body mass index (BMI) undergoing autologous or allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies were repeatedly shown to have higher rates of non-relapse mortality (NRM) and relapse, translating into a reduced overall survival (OS). However, the impact of obesity on outcomes after CAR T-cell therapy has not been studied at large scale. The present study aims to investigate whether high BMI is associated with outcomes in patients receiving anti-CD19 CAR T-cell therapy. Methods: This is a retrospective analysis using EBMT registry data of adult patients after first therapy with commercial CD19 CAR T-cell products for relapsed/refractory B-Non-Hodgkin lymphoma (B-NHL) between 1/1/2016 and 30/6/2022. Only patients with available information on height and weight at CAR T-cell therapy were included. Patients with a history of allogeneic stem cell transplantation were excluded. As we exclusively focused on the impact of increased BMI we excluded all patients with a BMI below 18.5 from the analysis. The primary outcome was the impact of BMI on OS. Multivariate analyses were performed with cause-specific Cox models, employing as risk factors patient age, sex, ECOG status and year of CAR T-cell therapy besides BMI categories (18.5-25, 25-30, >30). Results: A total of 4576 CAR T-cell therapy patients were registered during the study period, with 3125 patients meeting inclusion criteria. Selection of patients with available height and weight including BMI of 18.5 and above resulted in a final study cohort of 1653 patients. Median follow-up was 21.2 months [95% CI: 19.6-22.6]. Patients' characteristics are summarized in Table 1. Half of the cohort had a normal range BMI, while one third was overweight (BMI 25-30) and about 18% met criteria for obesity. A significant interaction was found between age and BMI. Therefore, we divided the cohort in two groups by the median age of 62.5 years. OS was significantly reduced in younger patients with normal range BMI compared to higher BMI patients. However, this effect was not significant in the older patients (Figure 1). Upon multivariate analysis, younger patients with BMI 25-30 had a survival advantage with a hazard ratio (HR) of 0.74 (0.57-0.95, P=0.02) and patients with BMI >30 a HR of 0.68 (0.51-0.91, P=0.01) compared to normal range BMI. Progression free survival was significantly improved for younger BMI 25-30 (HR 0.79, 0.64-0.99, P=0.04) but not BMI >30 (HR 0.82, 0.64-1.05, P=0.11), and NRM was reduced only in the younger BMI 25-30 subgroup (HR 0.52, 0.31-0.9, P=0.02) but not in the obese patients (HR 0.7, 0.42-1.18, P=0.19). Multivariate analysis of BMI subgroups in patients above the age of 62.5 years did not show significant differences with regard to OS (BMI 25-30 HR 0.94, 0.74-1.18; BMI >30 HR 1.04, 0.77-1.41), PFS (BMI 25-30 HR 1.06, 0.86-1.31; BMI >30 HR 1.1, 0.83-1.47), or NRM (BMI 25-30 HR 1.35, 0.89-2.06; BMI >30 HR 1.11, 0.6-2.06). Conclusion: Our European real world data on the impact of increased BMI on anti-CD19 CAR T-cell therapy for relapsed/refractory B-NHL shows that half of these patients are overweight or obese and that this seems to confer a survival benefit to younger (<62.5 years) patients. As this finding is rather surprising, future studies are required to fine tune the risk analysis, including in-depth analysis of patients' characteristics to identify protective factors linked to an elevated BMI as well as a possible selection bias. Our findings could in part be explained by the body weight-dependent dose of infused CAR T-cells for B-cell lymphoma. Therefore, for the same tumor burden, obese patients might receive more CAR T-cells than normal weight ones. A future study should test this hypothesis also in B-ALL or multiple myeloma patients, as they typically receive a fixed dose of CAR T-cells regardless of their body weight. Ultimately, our study might contribute to identify underlying mechanisms for improved outcome of CAR T-cell patients.

Is CAR T a Drug or a Therapeutic Pathway? Systematic Review, (Re-)Estimation of Efficacy Endpoints and Metanalyses in Large B-Cell Lymphoma Real World Reports

In everyday clinical practice, the therapeutic pathway of autologous CAR T-cell starts from leukapheresis, but the drug is infused on average 4-7 weeks later. Between leukapheresis and CAR T infusion patients may die following disease progression or exit the therapeutic pathway due to comorbidities or other reasons, thereby introducing a potential bias in the estimation of survival benefit gained with the drug. Moreover, substantial differences may occur (e.g., timing of leukapheresis and enrollment, use of bridging chemotherapy, lymphodepleting regimens) between trials' designs and every-day clinical practice. All of this precludes a robust comparison among the different available CAR T products and the therapies which share the same indication. Considering analyses limited to treated patients with endpoints estimated from date of infusion should be avoided to prevent misleading treatment choices. We conceived a project involving researchers and cooperative groups worldwide which already published real world effectiveness data on per-protocol (PP, i.e. from infusion) or on intention-to-treat (ITT, i.e. from leukapheresis taking into account also subjects who were scheduled for but finally did not receive CAR T) populations with the aim to re-estimate efficacy endpoints in both modalities. This would offer the clinicians the possibility to draw more meaningful interpretations of the data according to these two different approaches (ALMA IDEA 2022 CUP:J33C22001420001). First a systematic review was conducted searching for real world studies on the effectiveness of anti-CD19 CAR T-cell products administered to large B-cell lymphoma patients in third line and beyond between 2017 (after FDA approval) and October 2022. For each study, we asked researchers about both infused and not infused patients, for overall response rate (ORR), complete response rate (CRR), median follow up, median survivals (event-free [EFS], progression-free [PFS] and overall [OS]) duration of response (DoR). ITT EFS, the new endpoint added for our project, was defined as the time the patients remained in the CAR T pathway from leukapheresis to exit due to any cause. When applicable, meta-regressions, metanalyses and pooled analyses were also performed. The results overview with the 10 selected studies is reported in Table 1 (Bachy et al. Nat Med. 2022; Bastos-Oreiro et al. Front Immunol. 2022; Ghafouri et al. Clin Lymphoma Myeloma Leuk. 2021; Iacoboni et al. Cancer Med. 2021; Kittai et al. Transplant Cell Ther. 2021; Kwon et al. Haematologica. 2023; Mian et al. Leuk Lymphoma. 2021; Nastoupil et al. J Clin Oncol. 2020; Pinnix et al. Blood Adv. 2020; Casadei et al. Cancers (Basel). 2021). Rates of not infused patients ranged from 3% to 29%. Meta-regressions did not indicate a statistically significant influence of the different ratio of infused/not infused patients among studies, nor the different lengths of the median follow up seem to bias project results. Metanalyses for ITT ORR and CRR resulted in 57% and 38% respectively while the PP ORR and CRR were 68% and 45% respectively. Although centers kept track of all patients scheduled for CAR T, around 50% did not record the exit date (decision of not infuse, due to any cause) for not-infused subjects, reflecting the inability to calculate the ITT EFS. Three studies reported a median PP PFS shorter than the ITT PFS, which may indicate a high mean time from leukapheresis to infusion, worthy of further investigation. For all studies but one median PP OS was higher than ITT OS: OS may be over-estimated when using a PP analysis. We should be using an ITT analysis, as reporting data in which we do not include the patients who underwent leukapheresis and were never treated could introduce a significant bias in results interpretation. Hematologists should understand which is the most clinical meaningful endpoint for this new therapeutic approach to reach effective results: have we to consider CAR T-cell therapy as a drug or a therapeutic pathway? The importance of considering this new treatment option as a pathway should translate into implementation of CAR T patient registries that include ITT as an endpoint to establish population-level effectiveness of these drugs. In addition, considering both PP and ITT analyses as well as infused and not infused patients provides a complete overview and leads to more information.

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.

Prevention of CMV/EBV Reactivation By Double-Specific T Cells in Patients after Allogeneic Stem Cell Transplantation: Results from the Randomized Phase I/IIa Multivir-01 Study

Allogeneic stem cell transplantation (aSCT) is used to cure hematologic malignancies or deficiencies of the hematopoietic system. It is associated with severe immunodeficiency of the host early after transplant and therefore early reactivation of latent herpesviruses such as CMV and EBV within the first 100 days are frequent. Small studies and case series indicated that application of herpes virus specific T cells can control and prevent disease in this patient population. Study Design: A randomized, controlled, phase I/IIa multi-center study was conducted in 5 centers in Germany between 2013 and 2018 (ClinicalTrials.gov Identifier: NCT02227641). The study aimed at prevention or pre-emptive treatment of reactivation of CMV and EBV post aSCT. Major inclusion criteria were male or female HLA matched (10/10) unrelated or related donor seropositive for CMV and EBV, patient age between 18 and 75, the use of PBSC as stem cell source and the presence of on of the following HLA alleles: A*01:01, A*02:01, B07:02, B*08:01, B*35:01, C*07:02. CMV/EBV specific T cells were generated from a fraction of the allogeneic stem cell graft by stimulation with peptide pools for both viruses developed at the University Hospital Erlangen. Three doses of 5x10E4 CD3+ T cells per kilogram body weight were applied with an interval of at least 30 days without dose escalation starting earliest at day 30 after aSCT. Observation period was 204 days after aSCT. Primary endpoint of the study was toxicity of the T-cell-product assessed as acute transfusion reaction and acute GvHD due to the infusion of activated T cells. Results: 33 patients were screened and 29 patients were randomized. 16 patients were randomized into the treatment group of who 3 patients crossed over to the control arm due to failure of manufacturing the product or due to not meeting release criteria. Four patients were excluded from the study before receiving the first dose due to transplant related toxicity or withdrawal of consent. In total 9 patients were treated with 25 doses of the T cell product (dose range 2.1-4.1x10E4/kg CD3+ T cells). The earliest infusion was on day 30 (range: 30-69) post aSCT. There was no statistical difference between the groups regarding disease, patient age, donor type, performance status and comorbidities. The median follow up for both groups was 930 days. None of the patients treated experienced any acute transfusion reaction. Two patients developed acute GvHD after the second infusion and did not receive the third dose. Overall there was no statistical significant difference regarding survival (A) (treatment: 55.9% vs. control 69.2%, p=0.44), transplant related mortality (B) (22.2% vs. 7.1%, p=0.28), relapse (C) (11.1% vs. 14.3%) and acute GvHD (D,E) (33.3% vs. 46.2%, p=0.44). Number of AE per patient (27.1±24.6 vs. 24.4±11.2, p=0.60) and severity of AE (1.60±0.39 vs. 1.64±0.24) were similar in both groups. The incidence of reactivation of CMV (44.4% vs. 64.3%, p=0.16) and EBV (77.8% vs. 44.4%, p=0.12) was not statistically different and not associated with the treatment as several reactivations of occurred before application of the first dose. Within the treatment group we observed in several cases substantial expansion of CMV and EBV specific T cells in the peripheral blood which could not attributed to the presence viral antigens in case of reactivation. Conclusion: The study met its primary endpoint as there was no toxicity of the T-cell product observed. The cumulative incidence of acute GvHD as the primary endpoint was not statistically different in both groups and there was no acute toxicity related to the product. The study suffered from slow recruitment due to the market approval of Letermovir and low patient numbers did not allow for conclusive analysis of secondary endpoints such as the use of antiviral therapy.

Nex-T TM Cluster of Differentiation 19 (CD19) Chimeric Antigen Receptor (CAR) T-Cell Therapy BMS-986353 (CC-97540): Comparative Analysis of Baseline Patient Data from Trials in Systemic Lupus Erythematosus (SLE) and Relapsed/Refractory Large B-Cell Lymphoma (R/R LBCL) Related to Manufacturability and Potential Safety

Introduction: CAR T-cell therapy is currently being evaluated in non-oncologic autoimmune diseases (Mougiakakos D et al. N Engl J Med 2021; Müller F et al. Lancet 2023). In a cohort of patients (pts) with SLE, CD19 CAR T-cell therapy resulted in B-cell depletion and treatment-free, durable remission per Definitions of Remission In SLE (DORIS), suggesting that CD19 CAR T therapy may be feasible, well-tolerated, and effective for patients with SLE (Mackensen A et al. Nat Med 2022). Baseline traits associated with greater manufacturing success and lower toxicity profiles for CAR T have been identified in hematologic oncology (Mashadi-Hossein A et al. J Clin Oncol 2023; Rytlewski J et al. J Clin Oncol 2022) including age, bone marrow function, inflammatory state, and tumor burden. Here, we compared baseline traits in pts with active SLE versus R/R LBCL to predict the potential manufacturing success and toxicity profile for the ongoing phase 1 study to evaluate the investigational NEX-T TM CAR T-cell product BMS-986353 (CC-97540) in pts with severe, refractory SLE (NCT05869955). Methods: This post hoc analysis compared 42 quantitative baseline traits between pts with active SLE from PAISLEY (NCT03252587; Morand E et al. Arthritis Rheumatol 2023) and pts with R/R LBCL from TRANSCEND (NCT02631044; Abramson J et al. Lancet 2020). Baseline traits associated with improved manufacturing success and/or lower toxicity profile for CAR T in hematologic oncology were compared (Mashadi-Hossein A et al. J Clin Oncol 2023; Rytlewski J et al. J Clin Oncol 2022). A Wilcoxon rank-sum test was used to compare pt cohorts. Data are median (95% confidence interval). Results: This analysis included 363 pts with SLE and 256 pts with R/R LBCL. Of 42 baseline traits compared, 36 had nominal P values ≤ 0.05; 33 of those were ≤ 0.001. High baseline absolute lymphocyte count (ALC), platelets (PLT), and hemoglobin (Hgb) were previously associated with both improved manufacturing success and a lower toxicity profile for CAR T; pts with SLE had higher ALC, PLT, and Hgb vs R/R LBCL (ALC: 1.15 10 9/L [1.07-1.20 10 9/L] vs 0.10 10 9/L [0.10-0.13 10 9/L], P < 0.001, Figure 1A; PLT: 239 10 9/L [232-249 10 9/L] vs 144 10 9/L [129-154 10 9/L], P < 0.001; Hgb: 128 g/L [126-130 g/L] vs 98.0 g/L [95.0-100.0 g/L], P < 0.001), which is predictive of both improved manufacturing success and potentially lower toxicity. Young baseline age and high red blood cell (RBC) counts were previously associated with improved manufacturing success, and pts with SLE were younger and had higher RBC counts vs R/R LBCL (age: 40 years [38-41 years] vs 63 years [61-64 years], P < 0.001; RBCs: 4.30 10 12/L [4.20-4.40 10 12/L] vs 3.13 10 12/L [3.05-3.20 10 12/L], P < 0.001), predicting improved CAR T manufacturing success. Baseline traits reflecting tumor burden and/or bone marrow involvement that have been previously associated with higher toxicity, such as high C reactive protein (CRP; Figure 1B), high lactate dehydrogenase (LDH), and low hematocrit (Hct), were different between pts with SLE vs R/R LBCL (CRP: 1.92 mg/L [1.60-2.48 mg/L] vs 20.0 mg/L [15.5-31.8 mg/L], P < 0.001; LDH: 184 U/L [179-191 U/L] vs 282 U/L [251-317 U/L], P < 0.001; Hct: 39.0% [39.0-40.0%] vs 29.6% [28.7-30.5%], P < 0.001), and predict potentially lower toxicity. Conclusions: Comparison of baseline pt data from trials in SLE vs R/R LBCL suggests that pts with SLE have traits that predict improved manufacturing success and lower rates/severity of common CAR T-associated toxicities such as cytokine release syndrome (CRS) and neurological events. A key difference between cohorts is the potential for high tumor burden in R/R LBCL which has been previously associated with CAR T toxicity. Our results show marked differences between cohorts for tumor burden surrogate measures (CRP, LDH, Hct), bone marrow function, inflammatory state, and age which we hypothesize may potentially lead to improved CAR T manufacturing success and lower risk of CRS and/or neurological events for patients with autoimmune disorders vs R/R LBCL. Further research is needed to understand if CAR T-cell therapy may provide a promising treatment option for pts with SLE and other autoimmune conditions. The ongoing phase 1, multicenter, open-label study will evaluate the safety, feasibility, and preliminary efficacy of BMS-986353 (CC-97540) in patients with severe, refractory SLE. Study support: Bristol Myers Squibb

CD19-Directed CAR T-Cells (CD19-CAR) Combined with Acalabrutinib Achieves Durable Remissions in Patients with Relapsed or Refractory (r/r) Mantle Cell Lymphoma (MCL)

Background: MCL is an aggressive non-Hodgkin lymphoma (NHL) subtype which remains incurable despite improvements in progression free survival with consolidative autologous transplant and the introduction of novel targeted therapies [Dreyling et al. Blood 2012]. CD19-directed chimeric antigen receptor (CAR) T-cell therapy (CD19-CAR) has shown durable disease control in r/r MCL, but current standard of care products are limited by high rates of grade 3+ immune-related toxicities [Wang et al. NEJM 2020]. We hypothesized that combining a naïve/stem-memory (T N/SCM) phenotype-enriched CD19-CAR T-cell product with the selective BTK inhibitor (BTKi) acalabrutinib would attenuate the rates of severe toxicities, improve durable remission rates, and obviate the need for indefinite use of acalabrutinib in r/r MCL. Methods: We conducted a phase 1 single center, prospective clinical trial (NCT04484012) evaluating a single infusion of T N/SCM-enriched CD19.28.z.EGFRt-CAR at a target dose of either 2x10 8 (DL1) or 5x10 8 (DL2) CAR+ cells (≤20% lower dose allowed) in combination with ongoing acalabrutinib 100 mg twice daily for patients with r/r MCL after at least 1 prior line of therapy. Patients were required to undergo at least 3 months but less than 7 months of treatment with a BTKi prior to enrollment without evidence of progression and with measurable disease per Lugano criteria. All patients were switched to acalabrutinib for protocol therapy prior to lymphodepletion, and planned to continue until day 180 post CD19-CAR infusion at which time all therapy would be discontinued. We report outcomes from the dose finding portion of the study. Descriptive statistics were utilized for baseline characteristics and survival analyses were calculated utilizing the Kaplan-Meier method. Investigator-assessed response rates were based on Lugano criteria. Minimal residual disease (MRD) status was determined using either multiparameter flow cytometry (FCM) or next-generation sequencing (NGS). Results: To date, 8 patients enrolled and have received CD19-CAR (n=4 at DL1; n=4 at DL2). Manufacturing was successful in 100% of patients. Median time from leukapheresis to product infusion was 53 days (range, 41-114). Treated patients were 75% male; with a median age of 63 years (range, 38-70); and median number of prior therapies of 2 (range, 2-3). All patients had bone marrow involvement, and had high risk features including progression of disease within 24 months (n=6), relapse after autologous HCT (n=2), TP53 mutations (n=6), Ki-67 ≥30% (n=4), and/or complex karyotype (n=1). Prior to enrollment, all patients had received BTKi (n=8 acalabrutinib) for a median of 6.1 months (range, 4.5-7.3). No BTK and PLC-γ mutations were detected in patients with available samples. The combination of acalabrutinib and CD19-CAR was well tolerated; 5 patients (63%; n=1 grade 2; n=4 grade 1) had cytokine release syndrome (CRS), with no severe cases. No patients (0%) had immune effector cell associated neurotoxicity syndrome (ICANS) [Figure 1A]. No patient required corticosteroids for toxicity management, and only 2 patients (n=2/5, 40%) required tocilizumab for CRS. There were no serious adverse events (SAEs), and no treatment discontinuations due to AEs observed. The best ORR was 88% [n=6 CR (75%); n=1 PR (13%)], with median time to best response of 28 days. In those 6 patients who achieved CR, 2 patients were FCM-MRD negative (<10 -4) and 2 were NGS-MRD negative (<10 -6) (n=4/6, 67%) [Figure 1B]. After a median follow-up of 18.5 months (range, 8.3-28.9), 5 of 6 patients remain in CR, while1 patient who achieved an initial CR relapsed at 9.2 months. At 1-year post-infusion, the cumulative incidence of relapse and NRM were 30% (95% CI, 3%-67%) and 0%, respectively. The 1-year PFS and OS were 70% (95% CI: 22%-92%) and 100%, respectively. Conclusion: The combination of CD19-CAR with acalabrutinib was well tolerated and effective, yielding an ORR 88% and MRD- rate 50% in a cohort of patients with high-risk MCL. Notably, CRS events were manageable and low grade, and no ICANS was observed. Enrollment is ongoing in the expansion cohort (DL2), and additional clinical and correlative analyses will be presented at the meeting.

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.

Underrepresentation of Racial and Ethnic Minorities in Registrational Chimeric Antigen Receptor (CAR) T-Cell Therapy Trials in Hematologic Malignancies: An FDA Analysis

Introduction: Equitable representation of diverse racial and ethnic groups in oncology clinical trials continues to be a major challenge. Numerous initiatives designed to enhance diversity in oncology clinical trials are ongoing, including efforts at the U.S. Food and Drug Administration (FDA) such as the Oncology Center of Excellence's Project Equity. Given the multiple recent FDA approvals of CAR T-cell therapies for relapsed or refractory hematologic malignancies, we evaluated the enrollment rates of racial and ethnic groups in the CAR T-cell registrational trials submitted to the FDA. Methods: We conducted a pooled, patient-level analysis of all registrational trials of CAR T-cell therapies submitted to the FDA to support approval for hematologic malignancies between 2017 and 2021. Datasets from approved licensing applications were pooled, and demographics and efficacy outcomes were analyzed descriptively. Results: A total of 13 registrational trials involving 6 autologous CAR T-cell products were analyzed, comprising 1,319 patients with hematologic malignancies. The median age at enrollment was 60 years (range: 3 to 86 years). Of the 1,319 patients, 81% (1068) were White, 5% (66) were Black, 5% (65) were Asian, 4% (48) were reported as Other, and 8% (110) were Hispanic or Latino. Race and ethnicity information was not reported for 5% (72) and 10% (127) of patients, respectively. The majority of the patients enrolled in the registrational trials were from the United States (80%), followed by Europe (11%) and Australia (2%). The hematologic malignancies included large B cell lymphoma (59%), multiple myeloma (15%), follicular lymphoma (13%), B cell acute lymphoblastic leukemia (9%), and mantle cell lymphoma (5%). Response rates by race, ethnicity, and histology are shown in Table 1 and Table 2. Conclusions: Despite the majority of patients being enrolled in the U.S., racial/ethnic subgroups other than White and non-Hispanic/Latino were underrepresented in CAR T-cell registrational trials. This highlights the need to understand and overcome the continued barriers to CAR T-cell clinical trial enrollment across racial and ethnic groups. Although clinically meaningful efficacy was observed across these racial/ethnic groups and diseases, more data are needed to evaluate if efficacy outcomes vary across these subgroups. Work to describe safety and tolerability, particularly cytokine release syndrome (CRS) and neurotoxicity, in these racial and ethnic subgroups is ongoing. More effort is needed to better enhance diversity in CAR T-cell trials, especially given the expanding use of CAR T-cell therapies in the treatment of patients with hematologic malignancies.

Impact of Salvage and Bridging Therapy in Adult Patients with Refractory B-Cell Acute Lymphoblastic Leukemia (B-ALL) Referred for Anti-CD19 CAR T-Cell Therapy: An Intention to Treat Analysis

Background: Prognosis of relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL) is poor, particularly after allogeneic hematopoietic cell transplantation (alloHCT). In this setting, several CAR T-cell products targeting CD19 (CART19) have been able to achieve complete response (CR) rates of 70 to 90%. However, a significant proportion of these patients still relapse. Several studies focus on the effect of the CART19 product on efficacy, however, information on how salvage therapy (ST) and bridging therapy (BT) can modulate the outcomes of CART19 therapy is scarce. Methods: We report the outcomes of all adult patients with R/R B-ALL intended for treatment (aphaeresis performed) with available CART19 products in a single institution from Jul 2017 to Jul 2023. This includes patients intended to receive the academic varnimcabtagene autoleucel (var-cel) in the CART19-BE-01 trial and the consecutive compassionate use and hospital exemption programs, as well as with commercially available tisagenleuceucel (tisa-cel). Lymphodepletion was performed with fludarabine (90 or 120 mg/m 2) and cyclophosphamide (900 or 1000 mg/m 2), followed by the infusion var-cel or tisa-cel, respectively. We analyzed all ST (treatment administered after last relapse before CART19-apheresis) and BT (treatment administered after apheresis and before CART19 infusion) performed, and analyzed the impact of BT on PFS and OS after apheresis. Results: A total of 74 adult B-ALL patients underwent leukapheresis and CART19 (var-cel/tisa-cel) production. At screening patients had a median age of 34 years (19-78), 45% were females, with a median of 3 prior lines of therapy (2-7) including inotuzumab (69%), blinatumomab (27%) and allo-HCT (84%). After last relapse, 92% of patients received ST with a median of 1 (0-3) treatment line, including inotuzumab (48%), high-dose chemotherapy (28%), low-dose chemotherapy (21%), TKIs (12%), blinatumomab (9%), radiotherapy (7%) and DLIs (4%). The overall response to ST prior apheresis was MRD-negative CR (28%), MRD-positive CR (22%), partial response (4%) and progressive disease (PD) in 46%. After apheresis was secured, BT was performed in 76% of patients with a median of 1 (0-2) bridging line, including low-dose chemotherapy (44%), intrathecal therapy (23%), high-dose chemotherapy (15%), TKIs (15%), inotuzumab (14%), radiotherapy (3.5%), blinatumomab (1.8%) and DLIs (1.8%). BT was not administered in 24% (8/74) of patients mainly due to morphological remission at screening (83%) and frailty (11%). The overall response to BT prior CART19 infusion was MRD-negative CR (31%), MRD-positive CR (12%), partial response (7%) and progressive disease (47%), with a 3% treatment related mortality (TRM) events attributed to BT. Inotuzumab administration occurred either as ST or BT to CART19 in 86% of patients (the remaining 14% was previouly given as bridge to allo-HCT), and was able to achieve MRD-negative CR in 42% of patients. The overall control of the disease after BT either worsened (51%), remained similar (14%), or improved (35%). After this, 12% (n=9) of CART19 treatments were not performed due to progressive disease (33%), TRM (22%), therapeutic alternative (22%), and production failure (11%). Infused patients achieved an MRD-negative CRR of 90% with a median PFS of 11,2 months. There were no differences in OS between patients with ≥5% or <5% bone marrow (BM) blast counts either before apheresis (ITT, figure 1) or after BT prior to CART19 infusion. However, despite the lack of apparent impact of this disease burden categorization on the OS at both timepoints, a statistically significant difference was observed after categorizing patients regarding the response to BT (figure 2), with a median PFS of 6.6 vs 18.6 months if the overall disease status of the patients either worsened or remained stable/improved after BT (p=0.0002). Conclusions: To the best of our knowledge this could be one of the first studies focusing on the impact of ST and BT in adult patients with R/R B-ALL referred for CART19 therapy. These results suggest that the potentially synergistic impact of bridging therapy may be mediated more by the modulation exerted by BT on the proliferative kinetics of the disease rather than on the absolute tumour burden of the patient. More detailed results on the independent impact of each type of BT will be provided in the final presentation.

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 < 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.

Chimeric Antigen Receptor T Cell Therapies Clinical Trials in Pediatric Oncology: A Retrospective Analysis from Clinicaltrials.Gov

Background: Chimeric Antigen Receptor (CAR) T-cell clinical trials are growing exponentially. Database analysis can help identify scientific trends and generate projections regarding potential approvals before the results of the studies are completed or published. This is particularly interesting in pediatric oncology, where available analyses from repository databases are scarce or not available. Because of that reason, we sought to analyze the clinical trial public records from entries related to CAR T-cell clinical studies in pediatric patients (18 years or younger) uploaded to the clinicaltrials.gov database. Methods: We query the available data from clinicaltrials.gov using the following keywords: “CAR T cell in pediatrics”, “CAR T cell and cancer”, “chimeric antigen receptor”, “CAR T AND tumor antigen”, 'CAR T cell antigens“, ”Tumor antigens targeted by CAR T cells“, ”engineered T cells“, ”modified T cell“, ”CAR T cells in Cancer“, ”CAR T cell therapy“, ”CAR T cell therapy AND Cancer“ (data cut off July-2023). The data were tabulated, and the trials that did not meet the search criteria were excluded manually. Data cleansing was performed using published scientific data, web search-based information, and patent databases. Results: We found 4297 results in the Clinicaltrials.gov platform; we excluded 3978 clinical trials that did not involve the pediatric population (<18 years) or CAR T-cell intervention. The final analysis included 319 clinical trials posted on the clinicaltrials.gov website by the cut-off date. 207 (64.9%) were conducted in China, 74 (23.2%) in the US, and 21 (6.5%) in Europe. Most CAR T-cell trials focused on hematological cancer (89%), followed by non-CNS solid tumors 8%, and CNS tumors 3%. The most frequent targets were CD19 (46%), CD7 (7%), followed by GD2, CD22, CD123, CD19 + CD22 and CD30. Most clinical trials did not report the construct generation used in their CAR T cell product (58%). Among the trials with that information, second-generation constructs (2G) were used in 32% of the studies, followed by 3G and 4G constructs (5% of studies each). Most clinical trials were in early stages of development, Phase 1 or 2 (75%). We observed a continuous increase in the number of clinical trials registered yearly, except for a small reduction in 2022 and 2021, probably related to COVID restrictions and challenges. Overall, the general trend predicts at least a 50% yearly increase in registered studies ( Figure 1). Similarly, to what is observed in adult clinical trials, the most common signaling domains are 2G CAR constructs expressing either CD28 or 4-1BB as costimulatory domains. Among those, 4-1BB being is the most popular choice. Conclusion: Our findings indicate a growing interest in the clinical development of CAR T-cell therapies applied to pediatric oncology. Most of these studies are in early phase I and II and focus primarily on hematological cancers using second-generation constructs targeting CD19. The is a steady increase in the number of studies registered each year that predicts future FDA approvals using CAR T-cell products.

Seven-Day Vein-to-Vein Point-of-Care Manufactured CD19 CAR T Cells (GLPG5201) in Relapsed/Refractory CLL/SLL Including Richter's Transformation: Results from the Phase 1 Euplagia-1 Trial

Background Despite treatment advances, chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) remain incurable. Patients (pts) with concurrent Richter's transformation (RT) have poor prognoses with limited treatment options. Chimeric antigen receptor (CAR) T-cell products targeting CD19 have shown activity in pts with CLL/SLL or RT, yet data are limited and no therapies are approved. Due to limited treatment options and short survival of pts with RT, rapid treatment delivery is essential. A novel decentralized and automated point-of-care (PoC) manufacturing model was developed to administer fresh, autologous CAR-T treatments within 7 days of apheresis. An update on the Phase (Ph) 1 Euplagia-1 trial of PoC manufactured GLPG5201 is presented. Methods Euplagia-1 (CTIS: 2022-501686-47-00) is a Ph1/2 study of PoC-manufactured GLPG5201 in pts with R/R CLL or SLL, including pts with RT. Pts with CD19+ R/R CLL/SLL with ≥2 prior therapy lines are eligible. Pts with RT are eligible regardless of prior therapy. GLPG5201, an anti-CD19/41BB CAR-T therapy, is administered as a fresh product following fludarabine/cyclophosphamide lymphodepleting chemotherapy. Upon screening, pts receive daily ibrutinib until leukapheresis. The primary objectives are safety and determining a recommended Ph2 dose (Ph1), and objective response rate (ORR; Ph2). Secondary objectives include additional efficacy measures, pharmacokinetics and feasibility of PoC manufacturing. Results As of April 26, 2023, 12 pts were enrolled in Ph1 at dose level (DL) 1 (35×10 6 CAR+ T cells, n=6) or DL2 (100×10 6 CAR+ T cells, n=6) at a single institution. All pts were diagnosed with R/R CLL, 7/12 with concurrent RT. Median age was 66 years (range 58-71); 8/12 pts were male. Median prior lines of therapy were 4 (range 2-10); 10/12 pts received a BTK inhibitor, 9/12 pts venetoclax, and 1 pt an allogeneic stem-cell transplant. Five of 7 pts with RT relapsed after, or were refractory to, R-CHOP. Six of 12 pts had a TP53 mutation, 1 pt had a 17p deletion; 11 pts had an unmutated IGHV status and 2 had a complex karyotype reported. GLPG5201 was manufactured for all pts and administered as a fresh infusion. Median vein-to-vein time was 7 days (range 7-14). Due to lower manufactured CAR+ T cell yield, 3 pts received DL1 instead of the intended DL2. A preserved early memory phenotype for CD4+ and CD8+ CAR T cells was observed in the final product, compared with apheresis starting material ( Figure). Most treatment emergent adverse events (TEAEs) were Grade 1-2; most Grade ≥3 events were hematological ( Table). Six (50%) pts had Grade 1-2 cytokine release syndrome (CRS), with no CRS Grade ≥3. No neurotoxicity was reported. A dose limiting toxicity occurred in 1 pt (Grade 4 neutropenia, DL2), which was manageable and within expectations in this pt population. No unexpected GLPG5201-related toxicities were observed; no deaths occurred while pts were on study. Eleven pts responded to treatment (best ORR 92%). Complete response (CR) was observed for 9 pts (CR rate [CRR] 75%). ORR and CRR were 83% and 67% at DL1, and 100% and 83% at DL2, respectively. All but 1 pt with RT responded (ORR 86%); CR was achieved by 5/7 pts with RT (CRR 71%). One pt with RT was refractory with CD19-negative disease. At the time of analysis, 9/11 (82%) pts had ongoing responses; duration was up to 9 months post-infusion (median follow-up 3.2 months; range 1-9). Two pts progressed after an initial response, 1 pt with CD19-negative disease. Robust CAR T-cell expansion was observed in all pts by qPCR, with a median maximum expansion of 4.4×10 5 copies/µg DNA (C max). Median T max was 14 days (range 9-20 days). Median AUC 0-28d was 5.3×10 6 copies/µg DNA × days. GLPG5201 could be detected in peripheral blood up to 9 months post-infusion. Conclusion Data from 12 pts enrolled in the Ph1 Euplagia-1 study demonstrate that PoC CAR-T manufacturing with a short vein-to-vein time is feasible. GLPG5201 is administered as a fresh product; median vein-to-vein time is 7 days. There are no unexpected safety findings, and no CRS Grade ≥3 or any ICANS are reported. Efficacy data are encouraging, with a 92% best ORR and 75% CRR; 6/7 pts with RT responded. With this novel PoC manufacturing model, an early phenotype of less differentiated CAR T cells is preserved in the final product. GLPG5201 demonstrates rapid in vivo expansion and durable persistence post-infusion . Euplagia-1 is ongoing, and updated data will be presented.

CD27-Armored BCMA-CAR T Cell (CBG-002) Therapy for Relapsed and Refractory Multiple Myeloma: A Phase I Clinical Trial

Introduction: Several BCMA CAR T-cell products have been approved for relapsed or refractory multiple myeloma (RRMM), but long-term response has not been established. There is growing interest in the use of next-generation CAR T-cell therapy in RRMM. CBG002 is an armored BCMA CAR T-cell product engineered to constitutively express CD27, which binds CD70 to promote T-cell proliferation and differentiation. In vitro, CBG-002 cells exhibited enhanced proliferation, greater cytotoxicity and a higher proportion of memory T cells compared to control BCMA-CAR T cells; in animal models, CBG-002 infusion also resulted in improved tumor control and prolonged survival in MM xenograft mice. Based on the preclinical data, we conducted a phase I clinical trial to evaluate its safety and efficacy in patients with RRMM. Methods: This is a single-arm, open-label, phase I study (NCT04706936). Key eligibility criteria were RRMM patients who had received at least 3 prior regimens, including a proteasome inhibitor and an immunomodulatory drug; the percentage of BCMA expression ≥50% on myeloma cells, detected by either flow cytometry and immunohistochemistry. Patients received 3 days of fludarabine (30 mg/m 2) and cyclophosphamide (300 mg/m 2) followed by a single dose of CBG-002 infusion. The dose-escalation study follows a “3+3” design with 1×10 6/kg, 2×10 6/kg and 3×10 6/kg CAR-T cell cohorts. The primary endpoint was safety, including grade 3 or 4 treatment-emergent adverse events (TEAEs) and dose-limiting toxicities (DLTs); The secondary endpoints were overall response rate (ORR), duration of response (DOR), progression-free survival (PFS) and overall survival (OS). Results: As of June 25, 2023, 12 patients were enrolled and 1 patient was later excluded due to <50% BCMA expression. Among the evaluable 11 patients, there were 9 males and 2 females; median age was 52 years (range 36-66), 5/11 (45.5%) patients had high-risk cytogenetics including complex karyotype, t(4;14),1q21 amplification and del(17p), 4 (36.4%) had extramedullary disease (EMD). The median number of prior treatments was 5 (range 3-7); 7/11 pts were triple-agent exposed and refractory; 5/11 underwent ASCT. The most common TEAEs were neutropenia (G3/4 63.6%), thrombocytopenia (G3/4 36.4%) and anemia (G3/4 45.5%). Nine patients developed G1/2 CRS, which resolved spontaneously except for 2 patients who required steroids and tocilizumab, and no patients developed G3/4 CRS or ICANS. The median time to onset of CRS was 7 days (range 5-21) and the median duration was 6 days (range 5-8). At a median follow-up of 9 months, the ORR was 81.8%, including sCR/CR of 63.6%, VGPR 9.1% and PR 9.1%. After infusion, 8 pts were MRD negative in bone marrow by flow cytometry at day 28, 5 pts continued at month 3. The median OS was 14 months and the OS rate was 100%. The median PFS was 9 months and the PFS rate was 60%. The CBG-002 kinetics paralleled peripheral blood sCD27 levels with a median time to peak of 15 days (range 7-14). The fold increase in sCD27 levels was significantly higher in responders (12.1, range 1.2-83.8) than in non-responders (1.68, range 0.81-9.45). Conclusions: In this phase I study, CBG-002, a CD27-armored BCMA-CAR T therapy has demonstrated remarkable safety and clinical activity in RRMM patients.

CD123 As a Therapeutic Target in Accelerated and Blast Phase Myeloproliferative Neoplasms (MPNs)

Background: CD123 expression in MPNs has been reported in case series and CD123 has been a therapeutic target in one phase I study of patients with chronic myelomonocytic leukemia or myelofibrosis in chronic phase. CD123 has also been described as a marker of leukemic stem cells and incorporated in chimeric antigen receptor T-cell products for treatment of acute myeloid leukemia in clinical trials. Herein, we describe the clinical phenotype of patients with accelerated (AP) or blast phase (BP) MPNs paired with CD123 co-expression on CD45(dim) myeloid blasts. Methods: This is a retrospective, single-institution study of patients with AP and BP MPNs. Subjects were identified by search for MPN diagnoses in the Ohio State University Comprehensive Cancer Center Leukemia Tissue Bank (OSUCCC-LTB). Relevant clinical data was collected by retrospective chart review with approval of Institutional Review Board. CD123 expression was measured by immunophenotyping of cryopreserved peripheral blood (PB) or marrow (BM) samples. Blasts were identified by CD45(dim) gating of lymphocytes. Lymphocytes were also gated on CD7, CD13, CD33, CD34, and CD117. CD123 expression was stratified by quartile and correlation with disease phase, symptoms, splenomegaly, transfusion-dependence, and molecular mutations measured by Chi-square test. Results: Forty-one PB or BM samples from 24 subjects were identified from the OSUCCC-LTB. Nine subjects had paired PB and BM specimens and 6 subjects had samples obtained at multiple timepoints in the course of their disease. Median age at time of sample collection was 65.8 years (range 35.2-79.7) and median time from initial diagnosis to sampling was 13.1 months (range 0-96.5). Constitutional symptoms were present at the time of collection in 24 specimens (58.5%) and splenomegaly present in 20 specimens (51.3%). At sample collection, 19.5% had never received treatment, 19.5% had been previously treated, and 61% were currently on treatment. Most patients on treatment (n=16, 64%) were receiving JAK inhibitor at sample collection. Nineteen samples were obtained in chronic phase (46.3%), nine in AP (22.0%), and thirteen in BP (31.7%). Median WBC count was 4.37K/uL (range 0.3-174.6), median peripheral blasts were 3.2% (range 0-76%), median hemoglobin was 8.8 g/dL (range 5.6-13.2), and median platelets were 42K/uL (range 5-564). Sixteen samples (39.0%) were from transfusion-dependent patients, of which, 68.8% were both red blood cell (RBC) and platelet transfusion-dependent, 25.0% were RBC transfusion-dependent only, and 6.2% were platelet transfusion-dependent only. Median lactate dehydrogenase at time of sampling was 300 U/L (range 116-2,142). Median bone marrow blasts at time of sampling were 3% (range 0-82%). Molecular sequencing and karyotype +/- 30 days of sample collection were available for 18 (43.9%) and 27 (65.9%) of specimens, respectively. The most common mutations identified were JAK2 (50%), ASXL1 (27.8%), SRSF2 (22.2%), CALR (16.7%), and TP53 (16.7%). Normal karyotype was reported in 37% of the samples (one was consistent with donor chimerism post-allogeneic SCT), 33.3% had complex karyotype, 11.1% had del 5q or 7q/-7, and 18.5% had del 13q or 20q. CD123 expression on the CD45(dim) gated blasts ranged from 0.3-95.6% with a median value of 11.02%. CD123 expression is presented in figure 1 box plot. Figure 2a shows correlation between CD123 expression and AP/BP compared to chronic phase disease (p < 0.01). Figure 2b shows CD123 expression correlation with ASXL1 compared to unmutated ASXL1 (p =0.023). There were no statistically significant associations between CD123 expression and any other tested variables. Conclusions: These single-institution data suggest that high CD123 expression on blasts is associated with leukemic transformation and high-risk disease. This is demonstrated by correlation of higher expression in AP and BP MPN as well as correlation with ASXL1 mutation. CD123 expression in MPNs has been previously reported and explored as a therapeutic target in a single phase I trial, though these data suggest that specifically targeting high risk disease, defined by blasts >10% or the presence of ASXL1 mutation, may be more effective with greater target availability. This is an especially important subpopulation of patients with MPNs in need of more effective and less toxic therapy than current standards of care that typically include cytotoxic chemotherapy.

Chimeric Antigen Receptor (CAR) T-Cell Products for Pediatric Cancers: Why Alternative Development Paths Are Needed.

Chimeric Antigen Receptor (CAR) T-Cell Products for Pediatric Cancers: Why Alternative Development Paths Are Needed.

Immunosenescence and cancer: Opportunities and challenges.

As individuals age, cancer becomes increasingly common. This continually rising risk can be attributed to various interconnected factors that influence the body's susceptibility to cancer. Among these factors, the accumulation of senescent cells in tissues and the subsequent decline in immune cell function and proliferative potential are collectively referred to as immunosenescence. Reduced T-cell production, changes in secretory phenotypes, increased glycolysis, and the generation of reactive oxygen species are characteristics of immunosenescence that contribute to cancer susceptibility. In the tumor microenvironment, senescent immune cells may promote the growth and spread of tumors through multiple pathways, thereby affecting the effectiveness of immunotherapy. In recent years, immunosenescence has gained increasing attention due to its critical role in tumor development. However, our understanding of how immunosenescence specifically impacts cancer immunotherapy remains limited, primarily due to the underrepresentation of elderly patients in clinical trials. Furthermore, there are several age-related intervention methods, including metformin and rapamycin, which involve genetic and pharmaceutical approaches. This article aims to elucidate the defining characteristics of immunosenescence and its impact on malignant tumors and immunotherapy. We particularly focus on the future directions of cancer treatment, exploring the complex interplay between immunosenescence, cancer, and potential interventions.